Nucleotide sequences for regulating gene expression in plant trichomes and constructs and methods utilizing same

ABSTRACT

Provided is a novel plant regulatory sequence active in trichomes which is derived from the TR4 gene of wild tomato ( L. hirsutum ) and which comprises a nucleic acid at least 80% identical to SEQ ID NO:26. Also provided are nucleic acid constructs and methods of using same for directing expression of an exogenous polynucleotide sequences in trichomes and transgenic plant cells and transgenic plants which comprise the nucleic acid constructs.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 10/561,220, filed on Apr. 4, 2006, which is a National Phase ofPCT Patent Application PCT/IL2004/000549, filed on Jun. 20, 2004, whichclaims the benefit of U.S. Provisional Patent Application No.60/479,467, filed on Jun. 19, 2003.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to nucleotide sequences for regulatinggene expression in plant trichomes and, more particularly, to methods ofutilizing such nucleotide sequences for synthesizing polypeptides andmolecules of interest in plant trichomes.

Polypeptides can be expressed in a wide variety of cellular hosts. Foreconomic reasons, genetically engineered unicellular microorganisms aremost widely used for commercial production of polypeptides. However, insome cases, expression of mammalian proteins in unicellular organismsresults in incorrect folding and processing of the expressedpolypeptides leading to loss of biological or physiological activity ofthe obtained polypeptide. For these reasons, attempts have been made,with varying degrees of success, to express mammalian polypeptides inplants.

Transgenic plants are fast becoming a preferred system for theexpression of many recombinant proteins, especially those intended fortherapeutic purposes. One advantage of using plants is the potential forprotein production on an agricultural scale at an extremely competitivecost. Among other advantages is that most plant transformationtechniques result in a stable integration of the foreign DNA into theplant genome, so genetic recombination by crossing of transgenic plantsis a simple method for introducing new genes, accumulating multiplegenes into plants and avoiding the contamination of pathogens such asviruses and prions, which may affect human and animals. Furthermore, theprocessing and assembly of recombinant proteins in plants may alsocomplement that in mammalian cells, which may be an advantage over themore commonly used microbial expression systems.

Although plants provide a suitable alternative to unicellular expressionsystems, several disadvantages characterize current approaches forproduction of protein in plants. First, the concentration of theproduced protein is typically low (around 1% of total proteins) makingpurification extremely difficult. Second, other compounds may interferewith protein purification or even damage the proteins duringpurification. Third, expressing foreign proteins in propagated plantscan lead to environmental contamination and health risks associated withunwanted production of those proteins in cross pollinated plants.

In efforts of overcoming the above described limitations and whilereducing the present invention to practice, the present inventors havediscovered that plant trichomes enable compartmentalized production offoreign proteins as well as enzymatic production of novel chemicals,since many types of chemicals are naturally produced and even secretedfrom trichomes.

The above ground surfaces of many plants are covered with trichomes orhairs. The morphology of these structures can vary greatly with tissuetype and species. Indeed, the botanical literature contains more than300 descriptions (uniseriate, capitate-sessile, etc.) of variousmorphological types of such hairs (3, and references therein).Functionally, trichomes may be simple hairs that deter herbivores, guidethe path of pollinators, or affect photosynthesis, leaf temperature, orwater loss through increased light reflectance as in desert species.Alternatively, they may be more specialized tissues (glandular secretingtrichomes) whose principal function(s) may be to produce pest- orpollinator-interactive compounds that are stored or volatilized at theplant surface. It has been suggested that in some desert species theprincipal role of glandular secreting trichomes is to produce such highlevels of exudate that it forms a continuous layer on the plant surface.This layer may increase light reflectance and thereby reduce leaftemperature (30).

Trichomes develop projections from protodermal cells. Their structuresarise from a series of anticlinal and periclinal divisions to formsupporting auxiliary cells and glands. The appearance of glands atopsupporting cells and the occurrence of exudate around gland cells hassuggested that secretions are produced in gland cells and not by otherepidermal or subepidermal cells.

In several species, such as tomato and potato, a unique type oftrichomes accumulates certain protein (polyphenol oxidase) and compound(polyphenol) in the associated glands on the top of the trichome. Whenan insect lands on a leaf surface and contacts these trichomes, theydischarge their inner compounds thereby contacting the insect andsmearing it with a brown sticky compound, which is the product ofenzymatic oxidation of the polyphenols (reviewed in 4).

The mass production, accumulation, and secretion of such proteins andchemicals involve a specific genetic mechanism. This genetic mechanismincludes genes (5, 6) and promoters (7, 8, 9) acting in trichome cellsand cells organelles suited for accumulation and secretion of massproducts. This genetic mechanism allows, for example, trichome exudatesto reach 16% of total dry weight of leaves of a certain tobacco species(10) and a single protein to reach 60% of total proteins or aconcentration of 14 mg/mL in the trichome content of a solanum species(11, 12). The use of this genetic mechanism was suggested for tissuespecific production and accumulation of natural and heterologousproteins as well as chemicals (6). New compounds produced can bebeneficial for the plant itself by increasing resistance against pestssuch as insects, bacteria, and fungi (6), or for Molecular Farming orBio-Farming of human or mammalian proteins for the use as therapeutics.In the latter, harvesting the proteins produced in the trichomes can bemechanized.

Directing protein expression into trichome cells may involve the use ofpolynucleotides originated form different origins. A candidate sourcefor such regulatory elements is cotton as its fiber tissue isstructurally modified trichomes. The promoter sequences of cotton fiberspecific genes were shown to direct β-glucuronidase (GUS) expression tothe trichome cells of tobacco plants (7, 9). Alteration of trichomesstructure or chemistry by, for example, increasing cotton trichomelength or by producing pigments in the fiber could be beneficial for thecotton industry.

Natural chemicals of trichomes are already used as flavor, aroma,medicinals, pesticides, and cosmetic ingredients (13, 14). Naturalchemicals content was altered using antisense and co-suppression methods(6). However, enzymatic modifications of trichomes compounds via geneticengineering of genes, designed to produce other useful compounds intrichomes, was never shown before.

Several limitations had narrowed so far the use of plant trichomes forcommercially production of heterologous proteins and novel chemicals.

First, protein yield is very limited in trichome cells and to date thereis no existing method that enables commercially significant productionof proteins in these cells. Although there are known promoter sequencesthat are capable of directing protein synthesis in trichomes (7, 8, 9),proteins expressed therefrom accumulated at average levels ofaccumulation of a single trichome protein and thus these promoterscannot be considered commercially useful for protein production, as is.Second, trichomes usually produce a mix of several metabolites, some ofwhich (e.g., phenols and alkaloids), can inhibit protein accumulation orsubstantially hinder purification of desired compounds produced intrichomes (See material and methods in 12). Thus, reducing the levels ofsuch harmful metabolites is required in order to improve harvesting andcollection of the desired products. Third, the production of novelcompounds in plants always involves risks of escape of genetic material(pollen and seeds) to the environment with potential damage to otherorganisms (plants, insects animals, human). Hence, when producing novelcompounds one should consider the elimination of the possible spread ofthe new genetic material.

There is thus a widely recognized need for and it would be highlyadvantageous to have nucleotide sequences for regulating gene expressionin plant trichomes methods of utilizing such nucleotide sequences forgenerating molecules of interest in plant trichomes.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided anisolated polynucleotide comprising a nucleic acid sequence at least 80%identical to SEQ ID NO: 23, 26 or 29, wherein the nucleic acid sequenceis capable of regulating expression of at least one polynucleotidesequence operably linked thereto in trichomes.

According to another aspect of the present invention there is provided anucleic acid construct comprising the isolated polynucleotide.

According to further features in preferred embodiments of the inventiondescribed below, the nucleic acid construct further comprising at leastone heterologous polynucleotide operably linked to the isolatedpolynucleotide.

According to still further features in the described preferredembodiments the nucleic acid construct further comprises, a nucleic acidsequence encoding a peptide capable of directing transport of apolypeptide fused thereto into a subcellular compartment of a trichome.

According to still further features in the described preferredembodiments the nucleic acid sequence is selected from the groupconsisting of SEQ ID NOs: 59, 61, 63, 65 and 75.

According to yet another aspect of the present invention there isprovided a transgenic cell comprising the nucleic acid construct.

According to still another aspect of the present invention there isprovided a transgenic plant comprising the nucleic acid construct.

According to an additional aspect of the present invention there isprovided an isolated polynucleotide comprising a nucleic acid sequenceencoding a peptide capable of directing transport of a polypeptide fusedthereto into a subcellular compartment of a trichome, wherein thepeptide is encoded by the polynucleotide sequence set forth in SEQ IDNOs: 59, 61, 63, 65 and 75.

According to yet an additional aspect of the present invention there isprovided a nucleic acid construct comprising the isolatedpolynucleotide.

According to still further features in the described preferredembodiments the nucleic acid construct further comprising an expressiblepolynucleotide sequence translationally fused to the nucleic acidsequence encoding the peptide.

According to still an additional aspect of the present invention thereis provided a method of producing a polypeptide of interest in planttrichomes, the method comprising:

-   -   (a) expressing the polypeptide of interest in the plant        trichomes; and    -   (b) down-regulating a level of at least one molecule endogenous        to the plant trichomes, the at least one molecule being capable        of interfering with expression, accumulation or stability of the        polypeptide of interest.

According to still further features in the described preferredembodiments step (b) is effected by gene silencing.

According to a further aspect of the present invention there is provideda method of producing a molecule of interest in plant trichomes, themethod comprising: (a) expressing a polypeptide capable of directly orindirectly increasing a level of the molecule of interest in the planttrichomes; and (b) down-regulating a level of at least one moleculeendogenous to the plant trichomes, the at least one molecule beingcapable of interfering with accumulation or stability of the molecule ofinterest, thereby producing the molecule in the plant trichomes.

According to still further features in the described preferredembodiments the polypeptide is endogenously expressed in the planttrichomes.

According to still further features in the described preferredembodiments the expressing the polypeptide in the plant trichomes iseffected by introducing into the plant trichomes a nucleic acid sequenceencoding the polypeptide positioned under a transcriptional control of apromoter functional in the plant trichomes.

According to still further features in the described preferredembodiments the promoter is as set forth in SEQ ID NO: 23, 26, 29, 35,38, 39, 42, 48, 50 or 51.

According to still further features in the described preferredembodiments the nucleic acid sequence encoding the polypeptide ofinterest further encodes a peptide capable of directing transport of thepolypeptide fused thereto into a subcellular compartment of the planttrichomes.

According to still further features in the described preferredembodiments the at least one molecule is an enzyme or a metabolite.

According to still further features in the described preferredembodiments the metabolite is selected from the group consisting ofpolyphenols, ketones, terpenoids, phenylpropanoids and alkaloids.

According to still further features in the described preferredembodiments the enzyme is PPO.

According to still further features in the described preferredembodiments step (b) is effected by gene silencing.

According to yet a further aspect of the present invention there isprovided a plant genetically modified to express a molecule of interestin trichomes, wherein the plant is further modified or selected capableof accumulating less than 50% of average volume of undesired compoundsin trichome cells of the plant species.

According to still further features in the described preferredembodiments at least a portion of cells of the plant are geneticallymodified to include an expression construct including a polynucleotidesequence of a trichome specific promoter.

According to still further features in the described preferredembodiments the expression construct further includes an additionalpolynucleotide sequence encoding a peptide capable of directingtransport of a polypeptide fused thereto into a subcellular compartmentof the trichome, whereas the additional polynucleotide istranslationally fused to the polynucleotide sequence.

According to still further features in the described preferredembodiments at least a portion of cells of the plant are geneticallymodified to include an expression construct including a firstpolynucleotide sequence encoding the polypeptide translationally fusedto a second polynucleotide sequence encoding a peptide capable ofdirecting transport of a polypeptide fused thereto into a trichome.

According to still further features in the described preferredembodiments the expression or accumulation is in a subcellularcompartment of trichomes.

According to still further features in the described preferredembodiments the subcellular compartment is a leucoplast.

According to still further features in the described preferredembodiments the trichome specific promoter is set forth by SEQ ID NO:23, 26 or 29.

According to still further features in the described preferredembodiments the trichome specific promoter is set forth by SEQ ID NO:23, 26, 29, 35, 38, 39, 42 or 45.

According to still further features in the described preferredembodiments the additional polynucleotide sequence is set forth by SEQID NO: 59, 61, 63, 65 and 75.

According to still further features in the described preferredembodiments the molecule of interest is not a reporter polypeptide.

According to still further features in the described preferredembodiments the plant is modified or selected capable of generating atrichome density above 50,000 trichomes/gram (gr) leaf tissue.

According to still further features in the described preferredembodiments the plant is modified or selected capable of generating atrichome size of 50% above average size of the plant species.

According to still further features in the described preferredembodiments the plant is modified or selected capable of generating leafsurface size at least 25% above average size of the plant species.

According to still further features in the described preferredembodiments the plant is modified or selected capable of generatingtotal leaf number at least 50% above average leaf number of the plantspecies.

According to still further features in the described preferredembodiments the plant is sterile.

According to still further features in the described preferredembodiments the plant is further genetically modified capable ofsecreting the exogenous polypeptide from trichome cells.

According to still a further aspect of the present invention there isprovided a method of harvesting trichomes and/or exudates and/or contentthereof, the method comprising: (a) incubating a trichome-containingplant tissue in a liquid such that trichome exudates and content isreleased into the liquid, wherein incubating is effected while avoidingfriction of the trichome-containing plant tissue with a solid phase; and(b) collecting the liquid, to thereby harvest the trichome exudates andcontent.

According to still further features in the described preferredembodiments the liquid includes an antioxidant.

According to still further features in the described preferredembodiments the antioxidant is selected from the group consisting ofcitric acid, ascorbic acid and sodium bisulfite

According to still further features in the described preferredembodiments the liquid is water.

According to still further features in the described preferredembodiments the trichome-containing plant tissue is selected from thegroup consisting of a shoot, a flower and a leaf.

According to still a further aspect of the present invention there isprovided an apparatus for mechanical harvesting of trichome exudates andcontent, the apparatus comprising a mechanism designed and configuredfor mechanically agitating a trichome-containing plant tissue in a fluidand collecting the fluid to containing the trichome exudates or content.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing nucleotide sequence forregulating gene expression in plant trichomes and methods of utilizingsuch nucleotide sequences for generating molecules in plant trichomes

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor photograph. Copies of this patent or patent applicationpublication with color drawing(s) will be provided by the Office uponrequest and payment of the necessary fee.

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIG. 1 a is a prior art schematic illustration of various tomatotrichomes. Type VI glandular trichomes naturally accumulate high levelsof the PPO enzyme (Luckwill L C. 1943. The Aberden University Press,Aberden, Scotland).

FIGS. 1 b-h are photomicrographs depicting trichome specific expressionof GUS under the regulation of the CaMV 35S, TR2, TR5, TR11, TR25 orTR27P promoters. The blue color corresponds to GUS expression. FIG. 1b—Trichomes of wild-type tomato plants. FIG. 1 c—Trichomes of tomatoplants overexpressing GUS under the constitutive CaMV 35S promoter. FIG.1 d—Trichomes of tomato plants overexpressing GUS under the TR2promoter. FIG. 1 e—Trichomes of tomato plants overexpressing GUS underthe TR5 promoter. FIG. 1 f—Trichomes of tomato plants overexpressing GUSunder the TR11 promoter. FIG. 1 g—Trichomes of tomato plantsoverexpressing GUS under the TR25 promoter. FIG. 1 h—Trichomes of tomatoplants overexpressing GUS under the TR27 promoter.

FIG. 2 is a photograph depicting total protein yield of chemicallyextracted trichome cells as determined by coomassie staining.

FIG. 3 is a photograph depicting decreased PPO activity in the presenceof increasing concentrations of sodium bisulfite, as indicated by mediumbrowning, which is indicative of PPO activity.

FIG. 4 a schematically illustrates a trichome mechanical harvesterconstructed in accordance with some embodiments of the presentinvention.

FIG. 4 b schematically illustrates a trichome mechanical harvesterconstructed in accordance with some embodiments of the presentinvention.

FIG. 4 c schematically illustrates a trichome mechanical harvesterconstructed in accordance with some embodiments of the presentinvention.

FIGS. 5 a-c are graphs showing the effect of pruning on leaf number of 3tomato cultivars. FIG. 5 a cultivar 678; FIG. 5 b cultivar 1312; FIG. 5c cultivar 2545. Treatment 1—Plant shoot number was not limited, plantheight was limited to 1 m, flowers were cut-off before fruit set;Treatment 2—plant shoot number was not limited, plant height was limitedto 2 m, flowers were cut-off before fruit set; Treatment 3—plant shootnumber was not limited, leading apical meristem was cut (i.e. breakingapical dominance) when reached 0.5 m, flowers were cut-off before fruitset. Treatment 4—control plants were treated for tomato fruit set, suchthat each plant includes 2 shoots. Flowers and fruits were untouched.

FIGS. 6 a-d are graphs depicting expression levels of threetrichome-expressed genes (TR2H, TR4H and TR5H) as determined by RT-PCR.Expression is shown as fold increase over house-keeping gene expression.273_(—)1 is L. hirsutum var glabratum cultivar; 247 is L. esculentumcultivar. FIG. 6 a—a histogram depicting fold increase in the expressionlevel of TR2H in 273_(—)1 and 247 plants as compared to the expressionlevel in house-keeping genes; FIG. 6 b—a histogram depicting foldincrease in the expression level of TR2H in 247 plants as compared tothe expression level in house-keeping genes; FIG. 6 c—a histogramdepicting fold increase in the expression level of TR4H in 273_(—)1 and247 plants as compared to the expression level in house-keeping genes;FIG. 6 d—a histogram depicting fold increase in the expression level ofTR5H in 273_(—)1 and 247 plants as compared to the expression level inhouse-keeping genes; Tissue key: L-Leaves; L-T-Leaves minus Trichomes;T1 and T2 are two independent RNA samples of Trichome cells.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is nucleotide sequence for regulating geneexpression in plant trichomes which can be utilized for generatingmolecules in plant trichomes. Specifically the present invention is ofplants which are modified for enhanced expression and accumulation ofmolecules in plant trichomes.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

Trichomes are hairy-like epidermal multi-cell structures found on theouter surface of leaves, stems and flowers of about 20-30% of plantspecies. Their Main function is associated with plant protection againstinsects, microbes and herbivores due to their ability to massivelyaccumulate and secrete pest-deterrent phytochemicals. Other functionsinclude water absorption, seed dispersal and abrasion protection.

The high production capacity of trichomes prompted their utilization as“green-factories” for producing commercially useful compounds (e.g.,U.S. Pat. No. 6,730,826). However, mass production of recombinantproteins in trichomes is limited by poor production efficiency and thepresence of metabolites and enzymes, which may interfere withpurification and activity of the desired compounds.

While reducing the present invention to practice the present inventorshave devised a novel approach for increasing expression, accumulationand harvesting of molecules in plant trichomes, while reducing thechances of accidentally spreading the non-plant genetic material used togenerate the molecules.

As is illustrated in the Examples section, the present inventors haveuncovered that by reducing the concentration of undesired compounds intrichome exudates an enhanced level of expression, accumulation and/orpurification of commercially valuable molecules within the trichomes canbe achieved. Furthermore the present inventors uncovered throughlaborious experimentation and time consuming analysis a number of noveltrichome active regulatory elements (see Example 1), which enableprotein over-expression in trichomes (see Examples 2-4).

These findings allow, for the first time, improved molecular farming intrichome cells.

Thus, according to one aspect of the present invention there is provideda method of producing a molecule of interest in plant trichomes.

As used herein the term “trichome” refers to both a “simple” (alsotermed “non-glandular”) trichome and a “glandular-secreting” (GST)trichome. Preferably, the term trichome refers to a GST trichome.

As used herein the term “molecule” refers to at least one small moleculechemical (e.g., nicotine, flavonoids). Such molecules can be naturallyexpressed or present in trichomes or can be direct or indirectexpression products of heterologous polynucleotides. Examples ofmolecules which can be produced in trichome cells according to thisaspect of the present invention include, but are not limited to, oils,dyes, flavors, biofuels, or industrial biopolymers, pharmaceuticals,nutraceuticals and cosmeceuticals.

As used herein the term “producing” refers to the process of expressingand/or accumulating the molecule in trichome cells. When appropriate,producing may also refer to subsequent steps of purifying the moleculefrom the trichome cells.

The method, according to this aspect of the present invention, iseffected by upregulating expression of a polypeptide capable of directlyor indirectly increasing a level of the molecule of interest; anddown-regulating a level of at least one molecule endogenous to the planttrichome, which is capable of interfering with the production of themolecule in the plant trichomes, thereby producing the molecule in theplant trichomes.

Examples of polypeptides capable of directly or indirectly increasingthe level of the molecule of interest include for example, trichomespecific transcription factors which promote expression in trichomecells. Alternatively, the polypeptide can be an enzyme participating ina biochemical pathway, which produces the molecule in the trichome.

Expression of polypeptides in plant trichomes according to this aspectof the present invention, may be effected by placing a polynucleotideencoding the polypeptide of interest under the regulation of acis-acting regulatory element capable of directing expression from thepolynucleotide in trichome cells.

As used herein, the phrase “cis acting regulatory element” refers to apolynucleotide sequence, preferably a promoter, which binds a transacting regulator and regulates the transcription of a coding sequencelocated downstream thereto in trichome cells.

It will be appreciated that a regulatory sequence is “operably linked”to a coding polynucleotide sequence if it is capable of exerting aregulatory effect on the coding sequence linked thereto. Preferably, theregulatory sequence is positioned 1-500 bp upstream of the ATG codon ofthe coding nucleic acid sequence, although it will be appreciated thatregulatory sequences can also exert their effect when positionedelsewhere with respect to the coding nucleic acid sequence (e.g., withinan intron).

A number of trichome active promoters are known in the art which can beused in accordance with the present invention. Examples include, but arenot limited to, the CYP71D16 trichome-specific promoter [Wang E. J ExpBot. (2002) 53(376):1891-7, see U.S. Pat. No. 6,730,826] and the cottonLTP3 and LTP6 promoters (7, 9).

Methods of identifying trichome active or specific promoters are welldescribed in Examples 1-3 of the Examples section which follows.

As mentioned hereinabove, the present inventors have identified a numberof cis-acting regulatory elements, which are capable of regulatingtranscription of coding nucleic acid sequences operably linked theretoin trichome cells.

Thus the present invention provides an isolated polynucleotide having anucleic acid sequence at least 80% identical to SEQ ID NO: 23, 26 or 29,wherein the nucleic acid sequence is capable of regulating expression ofat least one polynucleotide sequence operably linked thereto intrichomes.

According to other embodiments of this aspect of the present inventionthe nucleic acid sequence of the present invention is at least 80%identical to SEQ ID NO: 35, 38, 39, 42, 45, 48, 51 or 54.

Preferably, the polynucleotides (promoters) of the present invention aremodified to create variations in the molecule sequences such as toenhance their promoting activities, using methods known in the art, suchas PCR-based DNA modification, or standard DNA mutagenesis techniques,or by chemically synthesizing the modified polynucleotides.

Accordingly, the sequences set forth in SEQ ID NOs: 23, 26, 29, 35, 38,39, 42, 45, 48, 51 and 54 may be truncated or deleted and still retainthe capacity of directing the transcription of an operably linked DNAsequence in trichomes. The minimal length of a promoter region can bedetermined by systematically removing sequences from the 5′ and 3′-endsof the isolated polynucleotide by standard techniques known in the art,including but not limited to removal of restriction enzyme fragments ordigestion with nucleases.

In another approach, novel hybrid promoters can be designed orengineered by a number of methods. Many promoters contain upstreamsequences which activate, enhance or define the strength and/orspecificity of the promoter, such as described, for example, by Atchison[Ann. Rev. Cell Biol. 4:127 (1988)]. T-DNA genes, for example contain“TATA” boxes defining the site of transcription initiation and otherupstream elements located upstream of the transcription initiation sitemodulate transcription levels [Gelvin In: Transgenic Plants (Kung, S.-D.and Us, R., eds, San Diego: Academic Press, pp. 49-87, (1988)]. Anotherchimeric promoter combined a trimer of the octopine synthase (ocs)activator to the mannopine synthase (mas) activator plus promoter andreported an increase in expression of a reporter gene [Min Ni et al.,The Plant Journal 7:661 (1995)]. The upstream regulatory sequences ofthe present invention can be used for the construction of such chimericor hybrid promoters. Methods for construction of variant promotersinclude, but are not limited to, combining control elements of differentpromoters or duplicating portions or regions of a promoter (see forexample, U.S. Pat. Nos. 5,110,732 and 5,097,025). Those of skill in theart are familiar with the specific conditions and procedures for theconstruction, manipulation and isolation of macromolecules (e.g., DNAmolecules, plasmids, etc.), generation of recombinant organisms and thescreening and isolation of genes, [see for example Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press,(1989); Mailga et al., Methods in Plant Molecular Biology, Cold SpringHarbor Press, (1995); Birren et al., Genome Analysis: volume 1,Analyzing DNA, (1997); volume 2, Detecting Genes, (1998); volume 3,Cloning Systems, (1999); and volume 4, Mapping Genomes, (1999), ColdSpring Harbor, N.Y].

The above-described nucleic acid sequences (promoters) can be used todrive expression of a heterologous polynucleotide of interest intrichome cells. Preferably, the heterologous polynucleotide can encodeany naturally occurring or man-made recombinant protein, such aspharmaceutical proteins [e.g., growth factors and antibodies SchillbergNaturwissenschaften. (2003) April; 90(4):145-55] and food additives. Itwill be appreciated that molecular farming is a well-proven way ofproducing a range of recombinant proteins, as described in details in MaNat Rev Genet. 2003 October; 4(10):794-805; Twyman Trends Biotechnol.2003 December; 21(12):570-8.

To facilitate accumulation of the polypeptide of interest in trichomecells, it may be beneficial to translationally link the heterologouspolynucleotide encoding the polypeptide to a signal peptide-encodingsequence which is capable of directing transport of the polypeptide intosub-cellular organelle of a the trichome. Examples of subcellularorganelles of trichome cells include, but are not limited to,leucoplasts, chloroplasts, chromoplasts, mitochondria, nuclei,peroxisomes, endoplasmic reticulum and vacuoles. Preferably the signalpeptide of this aspect of the present invention is a leucoplastlocalization signal. It is appreciated that since the protein is notaccumulated in the cytoplasm, but rather in the subcellular organelle ofthe trichomes, it is expected to be stored in relatively highconcentrations without being exposed to the degrading compounds presentin the trichome vacuole. Examples of signal peptides which may be usedin accordance with the present invention include, but are not limitedto, the stroma or lumen directing signal peptides of PPOA and PPOD (SEQID NO: 60, 62, 64, 66 and 76, see Example 3). Polynucleotides encodingthese signal peptides are set forth in SEQ ID NOs: 59, 61, 63, 65 and75.

The polynucleotides (i.e., trichome active promoter sequence, signalpeptide encoding polynucleotide) of the present invention, or fragments,variants or derivatives thereof, can be incorporated into nucleic acidconstructs, preferably expression constructs (i.e., expression vectors),which can be introduced and replicate in a plant cell, such as atrichome. Such nucleic acid constructs may include the heterologouspolynucleotide of interest such as described hereinabove, operably likedto any of the promoter sequences of the present invention.

The nucleic acid construct can be, for example, a plasmid, a bacmid, aphagemid, a cosmid, a phage, a virus or an artificial chromosome.Preferably, the nucleic acid construct of the present invention is aplasmid vector, more preferably a binary vector.

The phrase “binary vector” refers to an expression vector which carriesa modified T-region from Ti plasmid, allowing multiplication both in E.coli and in Agrobacterium cells, and usually comprising selectiongene(s). Such a binary vector suitable for the present invention isdescribed in Example 1 of the Examples section which follows.

The nucleic acid construct of the present invention can be utilized totransform a host cell. Preferably a plant cell. Preferably, the nucleicacid construct of the present invention is used to transform at least aportion of cells of a plant.

Methods of introducing nucleic acid constructs into a cell or a plantare well known in the art. Accordingly, suitable methods for introducingnucleic acid sequences into plants include, but are not limited to,bacterial infection, direct delivery of DNA (e.g., via PEG-mediatedtransformation, desiccation/inhibition-mediated DNA uptake,electroporation, agitation with silicon carbide fibers, and accelerationof DNA coated particles, such as described by Potrykus Ann. Rev. PlantPhysiol. Plant Mol. Biol. 42:205-225 (1991).

Methods for specifically transforming dicots primarily use Agrobacteriumtumefaciens. For example, transgenic plants reported include but are notlimited to cotton (U.S. Pat. Nos. 5,004,863, 5,159,135, 5,518,908; andWO 97/43430), soybean [U.S. Pat. Nos. 5,569,834, 5,416,011; McCabe etal., Bio/Technology, 6:923 (1988); and Christou et al., Plant Physiol.,87:671, (1988)]; Brassica (U.S. Pat. No. 5,463,174), and peanut [Chenget al., Plant Cell Rep., 15: 653, (1996)]. Similar methods have beenreported in the transformation of monocots. Transformation and plantregeneration using these methods have been described for a number ofcrops including but not limited to asparagus [Asparagus officinalis;Bytebier et al., Proc. Natl. Acad. Sci. U.S.A., 84: 5345, (1987); barley(Hordeum vulgarae; Wan and Lemaux, Plant Physiol., 104: 37, (1994)];maize [Zea mays; Rhodes, C. A., et al., Science, 240: 204, (1988);Gordon-Kamm, et al., Plant Cell, 2: 603, (1990); Fromm, et al.,Bio/Technology, 8: 833, (1990); Koziel, et al., Bio/Technology, 11: 194,(1993)]; oats [Avena sativa; Somers, et al., Bio/Technology, 10: 1589,(1992)]; orchardgrass [Dactylis glomerata; Horn, et al., Plant CellRep., 7: 469, (1988); rice [Oryza sativa, including indica and japonicavarieties, Toriyama, et al., Bio/Technology, 6: 10, (1988); Zhang, etal., Plant Cell Rep., 7: 379, (1988); Luo and Wu, Plant Mol. Biol. Rep.,6: 165, (1988); Zhang and Wu, Theor. Appl. Genet., 76: 835, (1988);Christou, et al., Bio/Technology, 9: 957, (1991); sorghum [Sorghumbicolor, Casas, A. M., et al., Proc. Natl. Acad. Sci. U.S.A., 90: 11212,(1993)]; sugar cane [Saccharum spp.; Bower and Birch, Plant J., 2: 409,(1992)]; tall fescue [Festuca arundinacea; Wang, Z. Y. et al.,Bio/Technology, 10: 691, (1992)]; turfgrass [Agrostis palustris; Zhonget al., Plant Cell Rep., 13: 1, (1993)]; wheat [Triticum aestivum; Vasilet al., Bio/Technology, 10: 667, (1992); Weeks T., et al., PlantPhysiol., 102: 1077, (1993); Becker, et al., Plant, J. 5: 299, (1994)],and alfalfa [Masoud, S. A., et al., Transgen. Res., 5: 313, (1996)]. Itis apparent to those of skill in the art that a number of transformationmethodologies can be used and modified for production of stabletransgenic plants from any number of target crops of interest.

The transformed plants can be analyzed for the expression featuresconferred by the polynucleotides of the present invention, using methodsknown in the art for the analysis of transformed plants (see Example 4of the Examples section which follows). A variety of methods are used toassess gene expression and determine if the introduced gene(s) isintegrated, functioning properly, and inherited as expected. Preferably,the promoters are evaluated by determining the expression levels and theexpression features of genes to which the promoters are operativelylinked. A preliminary assessment of promoter function can be determinedby a transient assay method using reporter genes, but a more definitivepromoter assessment can be determined from the analysis of stableplants. Methods for plant analysis include but are not limited toSouthern blots or northern blots, PCR-based approaches, biochemicalanalyses, phenotypic screening methods, field evaluations, andimmunodiagnostic assays. These methods may also be used to assess genesilencing, which is described hereinbelow.

As mentioned hereinabove, to enhance expression and/or accumulation ofthe molecule of interest in trichome cells and/or to facilitatepurification of the molecule from trichome cells, down-regulation of atleast one molecule endogenous to the plant trichomes and interferingwith these processes is effected.

Trichomes are known to include a number of compounds (e.g.,metabolites), which interfere with the production of molecules in thesespecialized cells. These metabolites include, for example polyphenols,ketones, terpenoids (e.g., monoterpenes, sesquiterpenes, diterpenes andtriterpenes), mixed terpenes, phenylpropanoids and alkaloids. Othertrichome components which may be preferably reduced to improve,expression, accumulation and purification of the molecules of thisaspect of the present invention include proteases, and PPO (see Example5 of the Examples section). For example downregulation PPO in trichomeplastids would allow the recruitment of the protein translationmachinery to a novel peptide and also to increase storage space intrichome plastids. Another example is reducing enzymatic activity of thepolyphenols biosynthetic pathway to thereby decrease/eliminate theproduction of polyphenols which make it difficult to harvest and purifyproteins from trichomes (see above). Such enzymes include, but are notlimited to, Phenylalanine ammonia-lyase (PAL, Acc. No. M90692, M83314),Cinnamate-4-hydroxylase (CA4H, GenBank Accession No. Z70216, A1490789),4-Coumarate:coenzyme A ligase (4CL, GenBank Accession Nos. AWO34240,AF211800), chalcone and stilbene synthase (CHS, Acc. No. GenBankAccession No. X55195), Chalcone isomerase (CHI, Acc. No. GenBankAccession No. AY348871), F3H, flavanone 3-hydroxylase-naringenin3-dioxygenase (F3OH), flavanone 3-hydroxylase-naringenin 3-dioxygenase(FDR), dihydroflavonol-4-reductase (DFR, GenBank Accession No. Z18277).

Down-regulation of such trichome components may be effected bydown-regulating genes which are involved in the production oraccumulation of these components. For example, gene products which areinvolved in exudate synthesis may be revealed by genome and EST miningand directed gene knock-out. Gene mining includes the identification inpublic databases (e.g., GenBank World Wide Web (dot) ncbi (dot) nlm(dot) nih (dot) gov/Genbank/index/html) of ortholohgous sequencesderiving from the plant of interest which share homology with knowngenes in the pathway using sequence alignment software such as BLAST(World Wide Web (dot) ncbi (dot) nlm (dot) nih (dot) gov/BLAST).Alternatively, trichome EST libraries may be useful for identifyinggenes which are involved in metabolite synthesis [see for example Lange(2000) Proc. Natl. Acad. Sci. 97:2934-2939; Gang (2001) Plant Physiology125:539-555].

Once genes associated directly or indirectly with metabolite synthesisare identified, they are down-regulated either at the nucleic acid leveland/or at the protein level (e.g., antibodies).

An agent capable of downregulating gene expression is a smallinterfering RNA (siRNA) molecule. RNA interference is a two stepprocess. the first step, which is termed as the initiation step, inputdsRNA is digested into 21-23 nucleotide (nt) small interfering RNAs(siRNA), probably by the action of Dicer, a member of the RNase IIIfamily of dsRNA-specific ribonucleases, which processes (cleaves) dsRNA(introduced directly or via a transgene or a virus) in an ATP-dependentmanner. Successive cleavage events degrade the RNA to 19-21 bp duplexes(siRNA), each with 2-nucleotide 3′ overhangs [Hutvagner and Zamore Curr.Opin. Genetics and Development 12:225-232 (2002); and Bernstein Nature409:363-366 (2001)].

In the effector step, the siRNA duplexes bind to a nuclease complex tofrom the RNA-induced silencing complex (RISC). An ATP-dependentunwinding of the siRNA duplex is required for activation of the RISC.The active RISC then targets the homologous transcript by base pairinginteractions and cleaves the mRNA into 12 nucleotide fragments from the3′ terminus of the siRNA [Hutvagner and Zamore Curr. Opin. Genetics andDevelopment 12:225-232 (2002); Hammond et al. (2001) Nat. Rev. Gen.2:110-119 (2001); and Sharp Genes. Dev. 15:485-90 (2001)]. Although themechanism of cleavage is still to be elucidated, research indicates thateach RISC contains a single siRNA and an RNase [Hutvagner and ZamoreCurr. Opin. Genetics and Development 12:225-232 (2002)].

Because of the remarkable potency of RNAi, an amplification step withinthe RNAi pathway has been suggested. Amplification could occur bycopying of the input dsRNAs which would generate more siRNAs, or byreplication of the siRNAs formed. Alternatively or additionally,amplification could be effected by multiple turnover events of the RISC[Hammond et al. Nat. Rev. Gen. 2:110-119 (2001), Sharp Genes. Dev.15:485-90 (2001); Hutvagner and Zamore Curr. Opin. Genetics andDevelopment 12:225-232 (2002)]. For more information on RNAi see thefollowing reviews Tuschl ChemBiochem. 2:239-245 (2001); Cullen Nat.Immunol. 3:597-599 (2002); and Brantl Biochem. Biophys. Act. 1575:15-25(2002).

Synthesis of RNAi molecules suitable for use with the present inventioncan be effected as follows. First, the mRNA target sequence is scanneddownstream of the AUG start codon for AA dinucleotide sequences.Occurrence of each AA and the 3′ adjacent 19 nucleotides is recorded aspotential siRNA target sites. Preferably, siRNA target sites areselected from the open reading frame, as untranslated regions (UTRs) arericher in regulatory protein binding sites. UTR-binding proteins and/ortranslation initiation complexes may interfere with binding of the siRNAendonuclease complex [Tuschl ChemBiochem. 2:239-245]. It will beappreciated though, that siRNAs directed at untranslated regions mayalso be effective, as demonstrated for GAPDH wherein siRNA directed atthe 5′ UTR mediated about 90% decrease in cellular GAPDH mRNA andcompletely abolished protein level (World Wide Web (dot) ambion (dot)com/techlib/tn/91/912 (dot) html).

Second, potential target sites are compared to an appropriate genomicdatabase (e.g., human, mouse, rat etc.) using any sequence alignmentsoftware, such as the BLAST software available from the NCBI server(World Wide Web (dot) ncbi (dot) nlm (dot) nih (dot) gov/BLAST/).Putative target sites which exhibit significant homology to other codingsequences are filtered out.

Qualifying target sequences are selected as template for siRNAsynthesis. Preferred sequences are those including low G/C content asthese have proven to be more effective in mediating gene silencing ascompared to those with G/C content higher than 55%. Several target sitesare preferably selected along the length of the target gene forevaluation. For better evaluation of the selected siRNAs, a negativecontrol is preferably used in conjunction. Negative control siRNApreferably include the same nucleotide composition as the siRNAs butlack significant homology to the genome. Thus, a scrambled nucleotidesequence of the siRNA is preferably used, provided it does not displayany significant homology to any other gene.

Antisense and siRNA technology has been used in selective downregulationof two tobacco trichome genes encoding different enzymes [Wang (2002);J. of Exp. Bot. 53:1891-1897; Wang (2003) Planta 216:686-691]. siRNAoligonucleotides for downregulating PPO for example, may be generated byinserting the cDNA sequence of PPO (GenBank Accession No: Z12833 forPPOA, GenBank Accession No. Z12836 for PPOD) to an siRNA selectionsoftware such as provided by World Wide Web (dot) ambion (dot) com.

Another agent capable of downregulating gene expression is a DNAzymemolecule capable of specifically cleaving an mRNA transcript or DNAsequence of the target. DNAzymes are single-stranded polynucleotideswhich are capable of cleaving both single and double stranded targetsequences (Breaker, R. R. and Joyce, G. Chemistry and Biology 1995;2:655; Santoro, S. W. & Joyce, G. F. Proc. Natl, Acad. Sci. USA 1997;943:4262) A general model (the “10-23” model) for the DNAzyme has beenproposed. “10-23” DNAzymes have a catalytic domain of 15deoxyribonucleotides, flanked by two substrate-recognition domains ofseven to nine deoxyribonucleotides each. This type of DNAzyme caneffectively cleave its substrate RNA at purine:pyrimidine junctions(Santoro, S. W. & Joyce, G. F. Proc. Natl, Acad. Sci. USA 199; for revof DNAzymes see Khachigian, L M [Curr Opin Mol Ther 4:119-21 (2002)].

Examples of construction and amplification of synthetic, engineeredDNAzymes recognizing single and double-stranded target cleavage siteshave been disclosed in U.S. Pat. No. 6,326,174 to Joyce et al.

Downregulation of gene expression can also be effected by using anantisense polynucleotide capable of specifically hybridizing with anmRNA transcript encoding the target polypeptide of interest.

Design of antisense molecules which can be used to efficiently andspecifically downregulate gene expression must be effected whileconsidering two aspects important to the antisense approach. The firstaspect is delivery of the oligonucleotide into the cytoplasm of theappropriate cells, while the second aspect is design of anoligonucleotide which specifically binds the designated mRNA withincells in a way which inhibits translation thereof.

In addition, algorithms for identifying those sequences with the highestpredicted binding affinity for their target mRNA based on athermodynamic cycle that accounts for the energetics of structuralalterations in both the target mRNA and the oligonucleotide are alsoavailable [see, for example, Walton et al. Biotechnol Bioeng 65: 1-9(1999)].

Such algorithms have been successfully used to implement an antisenseapproach in cells. For example, the algorithm developed by Walton et al.enabled scientists to successfully design antisense oligonucleotides forrabbit beta-globin (RBG) and mouse tumor necrosis factor-alpha (TNFalpha) transcripts. The same research group has more recently reportedthat the antisense activity of rationally selected oligonucleotidesagainst three model target mRNAs (human lactate dehydrogenase A and Band rat gp130) in cell culture as evaluated by a kinetic PCR techniqueproved effective in almost all cases, including tests against threedifferent targets in two cell types with phosphodiester andphosphorothioate oligonucleotide chemistries.

In addition, several approaches for designing and predicting efficiencyof specific oligonucleotides using an in vitro system were alsopublished (Matveeva et al., Nature Biotechnology 16: 1374-1375 (1998)].

Another agent capable of downregulating gene expression is a ribozymemolecule capable of specifically cleaving an mRNA transcript encodingthe target polypeptide. Ribozymes are being increasingly used for thesequence-specific inhibition of gene expression by the cleavage of mRNAsencoding proteins of interest [Welch et al., Curr Opin Biotechnol.9:486-96 (1998)]. The possibility of designing ribozymes to cleave anyspecific target RNA has rendered them valuable tools in both basicresearch and therapeutic applications.

An additional method of regulating the expression of a gene in cells isvia triplex forming oligonucleotides (TFOs). Recent studies have shownthat TFOs can be designed which can recognize and bind topolypurine/polypirimidine regions in double-stranded helical DNA in asequence-specific manner. These recognition rules are outlined by MaherIII, L. J., et al., Science, 1989; 245:725-730; Moser, H. E., et al.,Science, 1987; 238:645-630; Beal, P. A., et al, Science, 1992;251:1360-1363; Cooney, M., et al., Science, 1988; 241:456-459; andHogan, M. E., et al., EP Publication 375408. Modification of theoligonucleotides, such as the introduction of intercalators and backbonesubstitutions, and optimization of binding conditions (pH and cationconcentration) have aided in overcoming inherent obstacles to TFOactivity such as charge repulsion and instability, and it was recentlyshown that synthetic oligonucleotides can be targeted to specificsequences (for a recent review see Seidman and Glazer, J Clin Invest2003; 112:487-94).

In general, the triplex-forming oligonucleotide has the sequencecorrespondence:

oligo 3′--A G G T duplex 5′--A G C T duplex 3′--T C G A

However, it has been shown that the A-AT and G-GC triplets have thegreatest triple helical stability (Reither and Jeltsch, BMC Biochem,2002, Sep. 12, Epub). The same authors have demonstrated that TFOsdesigned according to the A-AT and G-GC rule do not form non-specifictriplexes, indicating that the triplex formation is indeed sequencespecific.

Thus, for any given sequence in the regulatory region a triplex formingsequence may be devised. Triplex-forming oligonucleotides preferably areat least 15, more preferably 25, still more preferably 30 or morenucleotides in length, up to 50 or 100 bp.

Transfection of cells (for example, via cationic liposomes) with TFOs,and formation of the triple helical structure with the target DNAinduces steric and functional changes, blocking transcription initiationand elongation, allowing the introduction of desired sequence changes inthe endogenous DNA and resulting in the specific downregulation of geneexpression. Examples of such suppression of gene expression in cellstreated with TFOs include knockout of episomal supFG1 and endogenousHPRT genes in mammalian cells (Vasquez et al., Nucl Acids Res. 1999;27:1176-81, and Puri, et al, J Biol Chem, 2001; 276:28991-98), and thesequence- and target specific down-regulation of expression of the Ets2transcription factor, important in prostate cancer etiology (Carbone, etal, Nucl Acid Res. 2003; 31:833-43), and the pro-inflammatory ICAM-1gene (Besch et al, J Biol Chem, 2002; 277:32473-79). In addition,Vuyisich and Beal have recently shown that sequence specific TFOs canbind to dsRNA, inhibiting activity of dsRNA-dependent enzymes such asRNA-dependent kinases (Vuyisich and Beal, Nuc. Acids Res 2000;28:2369-74).

Additionally, TFOs designed according to the abovementioned principlescan induce directed mutagenesis capable of effecting DNA repair, thusproviding both downregulation and upregulation of expression ofendogenous genes (Seidman and Glazer, J Clin Invest 2003; 112:487-94).Detailed description of the design, synthesis and administration ofeffective TFOs can be found in U.S. Patent Application Nos. 2003 017068and 2003 0096980 to Froehler et al, and 2002 0128218 and 2002 0123476 toEmanuele et al, and U.S. Pat. No. 5,721,138 to Lawn.

Regardless of the methods described hereinabove, the present inventionmay also be effected by using mutant plants or plant variants, which donot accumulate these metabolites or compounds (1, 16). Such plants canbe used for expressing and/or purifying the polypeptide of interest.Alternatively, such plants can be crossed with the transgenic plantsexpressing the polypeptide as described hereinabove. Next generationswill include plants, which both express the polypeptide of interest andproduce low levels of undesired compounds.

Plants generated or selected according to the above is preferablycapable of accumulating less than 50% of average volume of undesiredcompounds in the trichome cells of the pant species.

The present invention also envisages a method of producing a polypeptideof interest in plant trichomes. Such polypeptides can be endogenous tothe trichome or exogenous polypeptides, which can be used aspharmaceuticals (e.g., antibodies, antigens, ligands, growth factors,enzymes, structural proteins), industrial proteins and enzymes,therapeutics for veterinary use, proteins for molecular laboratories anddiagnostics, nutraceuticals or cosmeceuticals.

The method is effected by expressing the polypeptide in the planttrichomes, as described above, and down-regulating a level of at leastone molecule endogenous to the plant trichomes wherein such a moleculeis capable of interfering with the expression accumulation or stabilityof the polypeptide of interest.

Plants which may be utilized for trichome specific expression inaccordance with the present invention, are preferably selected orgenerated capable of generating (i) a trichome size of at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 80% aboveaverage size of the plant species; (ii) leaf surface size at least 5%,at least 10%, at least 15%, at least 25%, at least 30%, at least 40%above average size of the plant species; and/or (iii) total leaf numberat least 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 80% above average leaf number of the plant species; (iv) trichomedensity on the abaxial size of the leaf at least 10%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 80% above averagetrichome density of the plant species; (v) trichome density on theadaxial size of the leaf at least 100% above average trichome density ofthe plant species; (vi) shoot internode length of at least 5%, at least10%, at least 15%, at least 25%, at least 30%, at least 40% aboveaverage length of the plant species; (vi) trichome density above 50,000trichomes/gr leaf tissue; and/or (vii) trichome shape different thanthat of the plant species.

Plant architecture can be designed using genetic or non-geneticapproaches [Weston (1989) J. Amer. Soc. Hort. Sci. 114:492-498;Antonious (2001) J. Environ. Sci. Health B. 36(6):835-48].

A number of genes which are associated with trichome development andmorphogenesis were revealed through genetic studies. These genes mayregulate trichome initiation, division rate of trichome cells and/ortrichome cell ploidy number. A mutation in the TTG gene (GenBankAccession Nos. TTG1-AT5G24520, TTG2-AT2G37260) results in loss of leaftrichomes. Another example may be AGL16 (GenBank Accession No.NM_(—)115583), a recently discovered MADS-box gene that is expressed intrichomes [Alvarez-Buylla (2000) Plant J. 24:457-466]. Yet anotherexample is, KIC (GenBank Accession No. AY363866), a novel Ca²⁺ bindingprotein with one EF-hand motif, which interacts with a microtubule motorprotein and regulates trichome morphogenesis [Reddy Plant Cell. (2004)16:185-200]. Other genes, which affect trichome size and/ordistribution, include but are not limited to the UPL family of genes(e.g., UPL3, GenBank Accession No. AY265959), STICHEL [GenBank AccessionNo. AF264023, ligenfritz et. al. (2003) Plant Physiol. 131:643-55], COT1[Szymanski et. al. (1998) Genetics. 149:565-77], ZWICHEL (GenBankAccession No. AF002678, Oppenheimer et. al. (1997) Proc Natl Acad SciUSA. 10; 94:6261-6], GL1 (GenBank Accession No. AF263690), GL3 (GenBankAccession No. AT5G41315), GL2 (Acc. No. AT1G79840.1). It is conceivablethat such genes when over-expressed may increase trichome size and/ordistribution. For example, the GL-3 homologue R gene of maize causestrichome formation when over-expressed in Arabidopsis [Schellmann (2002)EMBO J. 21:5036-5046], indicating that such a manipulation is feasible.Overexpression of heterologous genes in plants is further detailedhereinbelow.

Non-transgenic approaches for modifying trichome size and/ordistribution include chemical or physical mutagenesis [Szymanski et. al.(1998) Plant Cell. 10:2047-62], somaclonal variation [Saieed et. al.(1994) Tree Physiol. 14:17-26; Guo et. al. (2003) Shi Yan Sheng Wu XueBao. 36:202-8] and induction of polyploidy [Melaragno et. al. (1993)Plant Cell. 5:1661-1668].

Trichome density can be increased by exposure to differentiating factors(i.e., non-genetic approaches). For example, day length (16).Alternatively, physiological concentrations of ethylene have been shownto promote trichome formation [Kazama (2001) Plant Physiol. 117:375-83].γ-radiation can be used to induce trichome formation [Negata (1999)Plant Physiol. 120:133-120].

Alternatively or additionally, trimming may be used to increase thenumber of leafs of the plant and as such increase the number oftrichomes (see Example 7 of the Examples section).

Plants of the present invention are preferably sterile (i.e., having noviable pollen or seeds) to prevent spreading of genetic material to thesurrounding environment. Sterilized plants can selected from mutantplants produced by for example chemical mutagenesis, physicalmutagenesis or by somaclonal variation. Alternatively sterilized plantscan be generated by silencing of fertility genes [Siaud et. al. (2004)EMBO J. 23:1392-401; Suzuki et. al. (2004) Plant J. 37:750-61; Li et.al. (2004) Plant Cell. 16:126-43; Krishnakumar and Oppenheimer (1999)Development. 126:3079-88].

Once plants are produced in accordance with the present invention,trichome content is purified to extract the molecules expressed thereinor the products thereof.

Mechanical and chemical methods of isolating trichomes and trichomesexudates and content are known in the art. Such methods include the useof solvent containing microcapillary for dissolving the exudates.Measures are taken, though, to select a solvent which does not interferewith the activity or stability of molecules thus purified. Anothermethod for removing trichomes include the use of forceps. A moreefficient method for isolating exudates is by washing the surface withan organic solvent. Again, measures are taken to select a solvent whichdoes not interfere with the activity or stability of molecules thuspurified. Trichomes may also be produced by brushing surfaces, shakingin an aqueous solution with an abrasive or freezing the tissue and thenbrushing [see McCaskill (1992) Planta 187:445-454; Wang (2001) NatureBiotechnology 19:371-374].

In order to facilitate collection of the trichome-produced molecule, thepresent inventors have devised a novel approach for large-scalecollection of trichome exudates and/or their content. This approach issimple to execute, does not require special technical skills, is costeffective and enables collection of large amounts of trichome exudatesand content.

Trichome content collection according to the present invention iseffected by incubating a trichome containing plant tissue in a liquid(e.g., water) such that trichome exudates and content is released intothe liquid. To avoid leaching of tissue components other than trichome,liquid incubation is effected while avoiding friction between thetrichome containing plant tissue and a solid surface. Thereafter, theliquid is collected, thereby harvesting the trichome exudates andcontent.

For example, trichome containing plant tissues, such as, shoots, leavesand flowers collected from plants can be incubated for 30-60 secondsunder agitation (60 times per minute) in water or any other liquid,which allows release of trichome content and exudates, while avoidingleaching of other tissue components. Preferably avoided are non-polarsolvents, such as chloroform and hexane.

The liquid is preferably supplemented with antioxidants such as citricacid, ascorbic acid and sodium bisulfite, which reduce the activity oftrichome components (e.g., PPO, see Example 5 of the Examples sectioninterfering with purification of the molecules.

Further purification of the molecules can be effected using any chemicalor biochemical method known in the art depending on the chemical natureof the molecule and its intended use. Such methods include, but are notlimited to, chromatography methods such as thin layer, affinity, gelfiltration and ion-exchange.

Collection of trichome exudates and content can be effected manually orby employing a collection apparatus specifically designed for such apurpose.

Thus, the present invention also envisages an apparatus for mechanicalharvesting of trichomes and/or trichome exudates and content(illustrated in FIG. 4 a), which is referred to herein as apparatus 10.

Apparatus 10 includes a collector 12 (e.g., brush, forceps, arm) whichis designed and configured for collecting trichomes and/or trichomeexudates and content from a trichome containing plant tissue 14.Accordingly, collector 12 includes a collecting mechanism 13 for holdingplant tissue 14 and a fluid filled reservoir 16 including fluid 18 inwhich plant tissue 14 is agitated by collecting mechanism 13.

Reservoir 16 also serves for storing collected trichomes and/or trichomeexudates and content.

As is illustrated in FIGS. 4 b-c, to enable agitation of plant material14 within the fluid of reservoir 16, apparatus 10 (collector 12)includes a vibrating mechanism 20 which is fitted with a motor or servoand a power unit either to collecting mechanism 13 (FIG. 4 b) or toreservoir 16 (FIG. 4 c). Apparatus 10 may also include an actuating unit22 and a timer 24 communicating with actuating unit 22. Reservoir 16 mayalso include at least one liquid channel 26 and pump 28 for transferringthe liquid with the trichomes out of the reservoir and into collectioncontainers or directly to a chromatography device for further separationand molecule isolation.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”,W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., Eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

Example 1 Cloning Promoter Regions of Trichome Expressed Genes andIdentifying Trichome-Active Promoters

Promoters suitable for expressing proteins in trichomes were identifiedby sequencing the genomic DNA upstream region of various cDNAs obtainedfrom genes expressed in leaf tissues.

Materials and Methods

Isolation and Cloning of Trichome Promoter Sequences in a Binary Vector:

The NCBI database of 126,000 tomato expressed sequence tags (ESTs)(including 5,000 ESTs originated from cDNA libraries originated from thetrichome tissues) and all transcribed nucleotide sequences described inliterature or directly submitted to NCBI (cDNAs) were used for theidentification of trichome active promoters. Keywords representing eachsequence and expression pattern thereof were collected and stored in adatabase.

LEADS™ software (Compugen, Ill.) was used for clustering and assemblingthe tomato sequences and provided more than 20,000 clusters representingdifferent genes. An expression profile annotative summary was designedfor each cluster by pooling all keywords of each sequence represented inthe cluster. Clusters were selected based on trichome EST number andpercentage out of total ESTs present in each cluster. Clusters wereanalyzed for ORFs using Vector NTI suite (InforMax, U.K.) version 6.ORFs of each gene were compared to GenBank database, using Blast(Hypertext Transfer Protocol://World Wide Web (dot) ncbi (dot) nlm (dot)nih (dot) gov/BLAST/) and for the highest homologous ORF the position ofthe ATG start codon and stop codon was compared. Accordingly, most ofthe sequences described herein were predicted to posses the full lengthORF. Clusters were classified as trichome-specific (i.e. more than 90%of ESTs in a cluster were originated from trichome cDNA libraries) ortrichome expressed (i.e. at least one of the ESTs in a cluster wasoriginated from trichome cDNA libraries).

RT-PCR—To verify the levels of expression and trichome specificityReverse Transcription following quantitative (Real-Time) PCR (RT-qPCR)was performed on total RNA extracted from either leaves, trichome cellsor leaves minus trichome cells.

mRNA levels were measured for three genes, previously predictedbioinformatically to express to high levels and specifically in trichomecells.

Trichome cells were harvested from tomato mature leaves by firstfreezing the leaves, just above liquid nitrogen and then brushing bothsides of the leaves with paint brush, previously chilled in liquidnitrogen. Total RNA was extracted from leaves, trichome cells or leavesminus trichome cells of tomato using RNEASY plant mini kit (Qiagen,Germany) using the protocol provided by the manufacturer. Reversetranscription was performed using 1.5 μg total RNA, using 300 U SuperScript II Reverse Transcriptase enzyme (Invitrogen), 225 ng randomdeoxynucleotide hexamers (Invitrogen), 500 μM dNTPs mix (Takara, Japan),0.2 volume of ×5 RT buffer (Invitrogen), 0.01M DTT, 60U RNASIN(Promega), DEPC treated DDW was added up to 37.5 μl.

RT reactions were incubated for 50 min at 42° C., followed by 70° C. for15 min. cDNA was diluted 1:20 in Tris EDTA, pH=8. 5 mL of the dilutedcDNA was used for qPCR.

To normalize the expression level between the different tissues specificprimers were designed for the following housekeeping genes: Actin (SEQID NO: 72), GAPDH (SEQ ID NO: 73), and RPL19 (SEQ ID NO: 74). Thefollowing primers were used for qPCR: Actin F primer:CCACATGCCATTCTCCGTCT (SEQ ID NO: 77), R primer GCTTTTCTTTCACGTCCCTGA(SEQ ID NO: 78); GADPH F primer TTGTTGTGGGTGTCAACGAGA (SEQ ID NO: 79), Rprimer ATGGCGTGGACAGTGGTCA (SEQ ID NO: 80); RPL19 F primerCACTCTGGATATGGTAAGCGTMGG (SEQ ID NO: 81), R primerTTCTTGGACTCCCTGTACTTACGA (SEQ ID NO: 82); TR2 F primertctcttcaattaggtacccgtcttg (SEQ ID NO: 83), R primerTGAATTTTGCCGTCATTGTCC (SEQ ID NO: 84); TR4 F primerGGGTTTAGACGTATCCGAAGGTC (SEQ ID NO: 85), R primerGCTCGTTTCCAATTTTCAGTAGAGA (SEQ ID NO: 86); TR5 F primerTTACGTGCCCAACTGAACACA (SEQ ID NO: 87), R primer CAATGCAATCAGCCCATGC (SEQID NO: 88).

qPCR was performed on cDNA (5 μL), using ×1 iQ™ SYBR Green super mix(BioRad), forward and reverse primers 0.3 μM each, and DDW was added upto 20 μL.

qPCR reaction was performed in iCycler real-time PCR machine (BioRad)95° C. for 3 min, 40 times of 95° C. for 15 sec and 1 min at 60° C.,followed by 95° C. for 15 sec, 60° C. for 60 sec, and 70 times of 60° C.for 10 sec+0.5° C. increase in each cycle.

The levels of expression (Qty) measured in the qPCR were calculatedusing the efficiency (E) of the amplification reaction and thecorresponding C.T. (the cycle at which the samples crossed thethreshold) Qty=E^(−C.T.). This calculation method assumes that theefficiencies of the reactions of the GOI (gene of interest) and of thehousekeeping genes are similar. In general the efficiencies of thereactions were 100%+/−5%.

Results are summarized in FIGS. 6 a-d.

FIGS. 6 a-d show that all three selected genes (i.e. TR2, TR4, and TR5)were expressed at high levels, up to 21 times higher than thehousekeeping genes, in trichome cells. In all cases expression washigher in trichomes compared to leaves, and in L. hirsutum compared toL. esculentum plants. Hence the promoter sequences, upstream to the genesequences, were cloned from L. hirsutum gDNA.

In order to clone these promoter sequences and 5′ untranslated region(5′ UTR) upstream of the ATG starting codon, total genomic DNA wasextracted from plant leaf tissues of 4 week old plants of the followingspecies: cultivated tomato (Lycopersicon esculentum, var 870), wildtomato species (Lycopersicon hirsutm, var LA 1777 and Lycopersiconpennellii, var LA 716), tobacco (Nicotiana tabaccum, var NN) or cotton(Gossypium hirsutum var Acala 23). DNA extraction was effected using DNAextraction kit (DNEASY plant mini kit, Qiagen, Germany). Inverse PCR(IPCR), DNA digestion, self-ligation, and PCR reaction were performed ongenomic DNA, following a well established protocol [Hypertext TransferProtocol://World Wide Web (dot) pmci (dot) unimelb (dot) edu (dot)au/core facilities/manual/mb390.asp] with the following modifications.To avoid mistakes in the IPCR, first the genomic sequence of the 5′sequence of a relevant cDNA (i.e. including introns) was identified toproduce Genomic Island (GI). The desired region from the genomic DNA wasPCR-amplified using direct oligonucleotide primers designed based on thecDNA cluster sequence, as was predicted by the Leads software (Compugen,Ill.). PCR reaction was performed in a DNA thermal cycler, using commonPCR conditions (for example: 92° C./3 min followed by 31 cycles×[94°C./30 sec; 56° C./30 sec; 72° C./3 min] followed by 72° C./10 min). PCRproducts were purified using PCR purification kit (Qiagen) andsequencing of the amplified PCR products was performed, using ABI 377sequencer (Amersham Biosciences Inc).

Primer sequences of each plant and the resultant GI sequence (i.e. thegenomic sequence which was amplified using the primers) are listed inTable 1, below.

TABLE 1 Forward primer/ Reverse primer/ Product size/ ID/PlantSEQ ID NO: SEQ ID NO: SEQ ID NO: TR2 (L. atggaagtaactttgttgtatagtac/GCCAGTGATCACCATAAGGAG/ 376/SEQ ID hirsutum) SEQ ID NO: 1 SEQ ID NO: 2NO: 3 TR4 (L. Ttctttggttcttcaatgttgg/ TTTGTAATGTCATTGGGAGGTC/410 bp of 5′ hirsutum) SEQ ID NO: 4 SEQ ID NO: 5 prime regionout of about 3500 bp of amplified PCR product SEQ ID NO: 6; Note−3500 bp were amplified by PCR, out of which only 5′ prime 410 bpwere sequenced TR5 (L. Gggtaatattcatttgattttcc/ AACCTGCTTTACATGTTTCAAG/431 bp/SEQ hirsutum) SEQ ID NO: 6 SEQ ID NO: 7 ID NO: 9

To increase amplification efficiency as needed a different amplificationtechnique [UP-PCR (20)] was employed. Briefly, UP-PCR technique was usedin order to amplify unknown upstream region of a known cluster sequence.Generally, the procedure involved four oligonucleotide primers: twosequence specific primers (SPs, external and internal) (listed below),both having the same orientation of 3′ end towards the unknown, yetdesired, 5′ region of the gene, and two universal walking primers (WP28and sWP). Reaction mixtures were generated as follows: sample mixture(SM)—genomic DNA of appropriate plant (tomato or cotton) species (30-40ng), WP28 primers (20 pmol), and DDW was added to a final volume of 10μl; Polymerase mixture (PM)—dNTPs (Roche, Switzerland) (10 mM each),Expand Long Template Enzyme mix (Roche, Switzerland) (1U), 10× buffersupplied with the enzyme and DDW was added to a final volume of 8 μl. SMwas placed in a thermocycler (Biometra, USA), where it was subjected toan amplification program of 1 minute at 90° C., held (pause) at 80° C.until PM was added, 30 seconds at 15° C., 10 minutes at 25° C., 3minutes at 68° C., held at 90° C. until the external SP (2 μl of 10 μMconcentration) was added. The process was followed by external PCRreaction of 30 seconds at 92° C., 10 seconds at 94° C., 30 seconds at65.5° C., 3 minutes at 68° C., for 30 cycles followed by final extensionof 10 minutes at 68° C.

External PCR products (diluted 5000-25000 fold) were used as templateand subjected to amplification using specific internal sWP and SP (30pmol each) primers, 1U Ex Taq (Takara), in 50 μl reaction volume.Internal PCR reactions were subjected to an amplification program of 2minutes at 92° C., followed by 30 seconds at 94° C., 30 seconds at 58°C., and 3 minutes at 72° C. for 30 cycles and a final extension of 10minutes at 72° C. IPCR/Up-PCR products were purified (PCR PurificationKit, Qiagen, Germany) and sequenced (ABI 377 sequencer, AmershamBiosciences Inc). Table 2, below, lists primers and products ofIPCR/Up-PCR reactions.

TABLE 2 External primers Internal primers SP sWP/ SP Product Amplifica-sWP28/ (external)/ SEQ ID (internal)/ SEQ ID ID/Plant tion methodSEQ ID NO: SEQ ID NO: NO: SEQ ID NO: NO: TR2/L. UP-PCR TTTTTTTTTGGAAGTTT TTTTTG GTGGGCTT 14 hirsutum TTGTTTGT AAGTAGTG TTTGTT GGTGGTAGTGTGGGG GGCTTG/SEQ  GTGGG/ ATTC/SEQ  GTGT/SEQ ID  ID NO: 11 SEQ IDID NO: 13 NO: 10 NO: 12 TR4/L. UP-PCR TTTTTTTTT GTTGAGTC TTTTTG CGAGCAGA17 hirsutum TTGTTTGT CACGAGCA TTTGTT CACTGTCA TGTGGGG GACAC/SEQ GTGGG/GAGG/SEQ GTGT/SEQ ID  ID NO: 15 SEQ ID ID NO: 16 NO: 10 NO: 12 TR5/L.UP-PCR TTTTTTTTT ATTCACAA TTTTTG GATGAGGT 20 hirsutum TTGTTTGT GGTTGTGGTTTGTT GTTTGGGT TGTGGGG ATGAGG/SEQ  GTGGG/ GCAC/SEQ ID GTGT/SEQ ID ID NO: 18 SEQ ID NO: 19 NO: 10 NO: 12

For cloning the putative promoters and 5′ UTRs, an additional PCRamplification was effected using a new set of primers (below) whichincluded 8-12 bp extensions having one restriction site (HindIII, SalI,XbaI, BamHI, or SmaI) on the 5′ prime end thereof. For each promoter,restriction sites that do not exist in the promoter sequence wereselected. Moreover, the restriction sites in the primer sequences weredesign such that the resultant PCR products were cloned into the binaryvector pPI in the right orientation, upstream of the GUS reporter gene.

The plasmid pPI was constructed by inserting a synthetic poly-(A) signalsequence, originating from pGL3 basic plasmid vector (Promega, Acc NoU47295; bp 4658-4811) into the HindIII restriction site of the binaryvector pBI101.3 (Clontech, Acc. No. U12640).

Table 3, below lists the restriction enzymes (enz.) containing primers,and the SEQ ID NO: of the resultant PCR products. Restriction siteswithin each primer are indicated by bold letters.

TABLE 3 Forward primer- Reverse primer- Product Restriction enz./Restriction enz./ SEQ ID ID/Plant SEQ ID NO: SEQ ID NO: NO: TR2(HindIII): (SalI): 23 (L. hirsutum) 5′-AATTTAAGCTTGTGTCG5′-AAATTGTCGACATCTCAA CTCAGCCCCTACTC-3′/ CTTGTTGCACTGAATTG-3′/SEQ ID NO: 21 SEQ ID NO: 22 TR4 (SalI): (BamHI): 26 (L. hirsutum)5′-CCTAGTCGACGGTGTTA 5′-TTGGATCCGAGCAGAC AATGGTGGGTTGG-3′/ACTGTCAGAGG-3′/ SEQ ID NO: 24 SEQ ID NO: 25 TR5 (HindIII): (BamHI): 29(L. hirsutum) 5′-TTTCCAAGCTTGACCTG 5′-CCGGATCCTCGTAAGGCTCTGATACCAATTG-3′/ AGTTTGTAATATG -3′/ SEQ ID NO: 27 SEQ ID NO: 28

PCR products were purified (PCR Purification Kit, Qiagen, Germany) anddigested with the restriction sites according to the primers used(Roche, Switzerland). The digested PCR products were re-purified andcloned into the binary vector pPI, which was digested with the samerestriction enzymes. PCR product and the linearized plasmid vector wereligated using T4 DNA ligase enzyme (Roche, Switzerland).

Example 2 Cloning of Trichome Active Promoter Sequences

Several genomic sequences were already described and validated in theliterature as trichome-specific promoters. In most cases promotervalidation was effected in tobacco plants. However, none of thesesequences were analyzed in tomato plants. Hence there is no way topredict which of these promoters will be active in tomato.

Materials, Methods and Results

A previously described tobacco promoter, (8) was isolated from genomicDNA (gDNA) of Nicotiana tabaccum, var Samsun NN using two sets ofoverlapping primers: 1. Forward—5′-AAATCTAGACTACCATCGCTAGTAATCGTG-3′(SEQ ID NO: 30) and Reverse—5′-GTTGAAGAACTGCATCCCGGGAGG-3′ (SEQ ID NO:31) to provide the sequence product set forth in SEQ ID NO: 32 (,TR25-2, SEQ ID NO: 67); 2.Forward—5′-AAATCTAGATAAGTTGATAAAGCTAATTTCTC-3′ (SEQ ID NO: 33) andReverse—5′-TTTCCCGGGACCTGGAGGCAATC-3′ (SEQ ID NO: 34) to provide thesequence product set forth in SEQ ID NO: 35 (TR25-3, SEQ ID NO: 68).

Primers sequences included additional restriction sites XbaI (Forwardprimers) and SmaI (Reverse primers), indicated in bold letters, tofacilitate further cloning.

Each PCR product was digested with XbaI and SmaI (Roche) and introducedvia ligation, using T4 DNA ligase (Roche), into pPI plasmid, digestedwith the same restriction enzymes.

A cotton promoter, previously described in (7), was isolated from gDNAof Gossypium hirsutum, var. Acala 23, and Gossypium barbadense var. Pima15 using the primers: Forward—5′-TATAAGCTTTAAGTTTAAATCCTATTGTAGTG-3′(SEQ ID NO: 36) and Reverse—5′-CGGATCCATTAATCACAAGAAAAAC-3′ (SEQ ID NO:37) to provide a genomic amplified sequence of Acala as set forth in SEQID NO: 38 (27A) and a genomic amplified sequence of Pima as set forth inSEQ ID NO: 39 (27P).

Primer sequences included additional restriction sites HindIII (Forwardprimer) and BamHI (Reverse primer), indicated in bold letters, tofacilitate further cloning. PCR products were digested with HindIII andBamHI (Roche) and introduced via ligation, using T4 DNA ligase (Roche),into pPI plasmid, digested with the same restriction enzymes.

Example 3 Cloning of Tomato PPO Promoters and Signal Peptide

Tomato polyphenol oxidase (PPO, GenBank Accession No: Z12833 for PPOA,GenBank Accession No. Z12836 for PPOD) is the major protein in type VItrichomes of tomato (5). Hence it was expected that the promoter regionupstream the PPO gene will direct the expression of foreign genes to thetrichome cells. PPO is encoded by closely related, seven members, genefamily. A previous publication identified which of the gene familymembers are preferably expressed in the trichome cells (5).

The genomic sequence of the PPO gene family was published. Still, inmost cases, promoter activity was not tested for the sequences upstreamof the genes.

Materials, Methods and Results

The promoter sequence of PPOA and PPOD was cloned from wild tomato(Lycopersicon pennellii) and cultivated tomato (Lycopersiconesculentum), respectively. Cloning of the putative promoter region ofPPOA was effected by amplifying the genomic sequence upstream of thecoding region, using the primers:

Forward—5′-AAAATTTGGGATCTAGAAGGTGAGG-3′ (SEQ ID NO: 40) andReverse—5′-CTGGATCCTATTGCTAGCTTTGGATGAAG-3′ (SEQ ID NO: 41). Theresultant genomic DNA amplified thereby is set forth in SEQ ID NO: 42(T8). Primer sequences include additional restriction sites XbaI(Forward primer) and BamHI (Reverse primer), indicated in bold, tofacilitate further cloning.

The resultant PCR product was digested with XbaI and BamHI (Roche) andintroduced via ligation, using T4 DNA ligase (Roche), into pPI plasmid,digested with the same restriction enzymes.

Cloning of the putative promoter region of PPOD was performed byamplifying the genomic sequence upstream of the coding region, using theprimers: Forward—5′-ATGGAAAAGCTTATGGACAGACTAAAACAC-3′ (SEQ ID NO: 43)and Reverse—5′-CTGGATCCTGTTGCTAGCTTTTGAATGAAA-3′ (SEQ ID NO: 44). Theresultant genomic DNA amplified thereby is set forth in SEQ ID NO: 45(T11).

Primer sequences included additional restriction sites HindIII (Forwardprimer) and BamHI (Reverse primer), indicated in bold, to facilitatefurther cloning.

PCR product was digested with HindIII and BamHI (Roche) and introducedvia ligation, using T4 DNA ligase (Roche), into pPI plasmid, digestedwith the same restriction enzymes.

Vast accumulation of PPO in trichomes is largely governed by proteinimport and storage within the thylakoid lumen of plastids, such aschloroplasts and leucoplasts (5, 12, 28). Protein import into the lumenis directed by a signal peptide on the amino terminal of the immaturepolypeptide of PPO. The immature polypeptide is imported first into theplastid stroma where the primary part of the signal peptide is cleaved.Later on, the second part of the signal peptide is cleaved, while thepolypeptide is crossing the thylakoid membrane and the maturepolypeptide is entering into the thylakoid lumen (28).

Hence to facilitate the accumulation of foreign proteins in trichomes,protein import into plastids might be crucial.

To test protein import, the native signal peptide of either PPOA or PPODwas amplified together with the putative promoter of each gene.Amplified products were cloned into pPI and fused, in frame, to the GUSreporter gene. Amplification of PPOA promoter together with only theinitial part of the signal peptide, which directs protein to the stromawas done using the following primers:Forward—5′-AAAATTTGGGATCTAGAAGGTGAGG-3′(SEQ ID NO: 46, XbaI restrictionsite is indicated in bold) and Reverse—5′-ACATGAAACTTTGAATGCTTTG-3′ (SEQID NO: 47). The genomic amplified sequence of PPOA is set forth in SEQID NO: 48.

PCR product was digested with XbaI (Roche) and introduced via ligation,using T4 DNA ligase (Roche), into pPI plasmid, digested with XbaI andSmaI restriction enzymes.

Amplification of PPOD promoter and signal peptide which directs proteinto the stroma was effected using the following primers:Forward—5′-ATGGAAAAGCTTATGGACAGACTAAAACAC-3′ (SEQ ID NO: 49) andReverse—5′-TTCCCGGGACATGAAACTTTGAATGCTTTG-3′ (SEQ ID NO: 50). Genomicamplified sequence of PPOD is set forth in SEQ ID NO: 51.

Primer sequences included additional restriction sites HindIII (Forwardprimer) and SmaI (Reverse primer) to facilitate further cloning.

PCR product was digested with HindIII and SmaI (Roche) and introducedvia ligation, using T4 DNA ligase (Roche), into pPI plasmid, digestedwith the same restriction enzymes.

Amplification of PPOD promoter and signal peptide which directs proteinto the lumen was done using the following primers:Forward—5′-ATGGAAAAGCTTATGGACAGACTAAAACAC-3′ (SEQ ID NO: 52) andReverse—5′-AACCCGGGAGCCGATGCAGCTAATGG-3′ (SEQ ID NO: 53). The resultantgenomic amplified sequence of PPOD is set forth in SEQ ID NO: 54.

Primer sequences included additional restriction sites HindIII (Forwardprimer) and SmaI (Reverse primer), indicated in bold, to facilitatefurther cloning.

PCR product was digested with HindIII and SmaI (Roche) and introducedvia ligation, using T4 DNA ligase (Roche), into pPI plasmid, digestedwith the same restriction enzymes.

Example 4 Expression of Therapeutic Proteins in Trichome Cells

Materials and Methods

The potential of trichomes to accumulate human therapeutic proteins canbe estimated by expressing human interferon β gene or human growthhormone gene in trichome cells.

Materials and Methods

Cloning of human interferon β into a binary vector—The gene for humaninterferon β (INFB, GenBank Accession No. NM_(—)002176) was amplifiedfrom human genomic DNA using the following primers: Forward:5′-GGGATGAGCTACAACTTGCTTGGAT-3′ (SEQ ID NO: 55) and Reverse:5′-CTAGGAGCTCTTCAGTTTCGGAG-3′ (SEQ ID NO: 56, a SacI restriction site onthe primer is indicated in bold). The resultant sequence of the INFBgene is set forth in SEQ ID NO: 57.

Analysis of the INFB sequence revealed a codon usage that is similar tothe codon usage of the tomato (data not shown).

The PCR product (SEQ ID NO: 57) was digested using SacI restrictionendonuclease (Roche) and cloned into pPI binary vector digested withSmaI and SacI, hence replacing the GUS gene. The newly formed binaryplasmid was designated pINFB. Sequence analysis of the INFB gene inpINFB revealed that the INFB sequence was cloned in the rightorientation. Trichome promoters together or without a plastid transitpeptides (summarized in Table 4 below) were further cloned upstream ofthe INFB gene in pINFB.

Cloning of human growth hormone into a binary vector—The maturepolypeptide of the Human-growth hormone gene (HGH, GenBank Accession No:V00519) was produced synthetically using GeneArt service (HypertextTransfer Protocol://World Wide Web (dot) geneart (dot) de/). Thesequence was adjusted according to the tomato codon usage, whileavoiding, as much as possible, high GC content and low complexity of DNAsequences. An ATG was added as a first codon to the mature polypeptideenabling sufficient translation. The restriction sites of SmaI and SacIwere added to the gene at the 5′ prime end and 3′ prime end,respectively. The sequence of the HGH gene is set forth in SEQ ID NO:58.

The gene clone was provided in a PCR script plasmid vector. The gene wasdigested out of the plasmid using SmaI and SacI restriction endonucleas(Roche) and cloned into pPI binary vector, replacing the GUS gene. Thenewly formed binary plasmid was named pHGH. Sequence analysis revealedthat the inserted HGH gene in pHGH was cloned in the right orientation.Trichome promoters with or without a plastid transit peptides(summarized in Table 4, below) were further cloned upstream of the HGHgene in pHGH.

Agrobacterium transformation of binary plasmids expressing heterologousgenes—Agrobacterium tumefaciens (strains LBA4404) competent cells weretransformed with 0.5 μl binary plasmid by electroporation, using aMicroPulser electroporator (Biorad, USA), 0.2 cm cuvettes (Biorad, USA)and EC-2 electroporation program (Biorad, USA). Cells were incubated inLB medium at 28° C. for 3 hours and plated on LB-agar platessupplemented with 50 mg/L kanamycin (Sigma, USA) and 250 mg/Lstreptomycin. Plates were incubated at 28° C. for 48 hours untilAgrobacterium colonies grew. These colonies were subsequently used fortobacco or tomato plant transformation.

Plant transformation and cultivation—Table 4, below, summarizes theconstructs which were introduced into tomato plants.

TABLE 4 Promoter species Transit peptide gene binary 35S CaMV No GUS pPITR2 L. hirsutum No GUS pPI TR4 L. hirsutum No GUS pPI TR5 L. hirsutum NoGUS pPI TR8 L. pennellii No GUS pPI TR8 L. pennellii Stroma GUS pPI TR11L. esculentum No GUS pPI TR11 L. esculentum Stroma GUS pPI TR11 L.esculentum Lumen GUS pPI TR25-2 N. tabaccum No GUS pPI TR25-3 N.tabaccum No GUS pPI TR27-A G. hirsutum No GUS pPI TR27-P G. barbadenseNo GUS pPI TR2 L. hirsutum No INFB pINFB TR4 L. hirsutum No INFB pINFBTR5 L. hirsutum No INFB pINFB TR8 L. pennellii No INFB pINFB TR8 L.pennellii Stroma INFB pINFB TR11 L. esculentum No INFB pINFB TR11 L.esculentum Stroma INFB pINFB TR11 L. esculentum Lumen INFB pINFB TR25-2N. tabaccum No INFB pINFB TR25-3 N. tabaccum No INFB pINFB TR27-A G.hirsutum No INFB pINFB TR27-P G. barbadense No INFB pINFB TR2 L.hirsutum No HGH pHGH TR4 L. hirsutum No HGH pHGH TR5 L. hirsutum No HGHpHGH TR8 L. pennellii No HGH pHGH TR8 L. pennellii Stroma HGH pHGH TR11L. esculentum No HGH pHGH TR11 L. esculentum Stroma HGH pHGH TR11 L.esculentum Lumen HGH pHGH TR25-2 N. tabaccum No HGH pHGH TR25-3 N.tabaccum No HGH pHGH TR27-A G. hirsutum No HGH pHGH TR27-P G. barbadenseNo HGH pHGH

Tomato transformation—Tomato transformation was carried out according toFillati et al. (19). Briefly, Lycopersicon esculentum cv. Micro-Tomcotyledons were used for Agrobacterium based plant transformation. TheMicro-Tom seeds were surface sterilized for 10 min in a 3% sodiumhypochlorite solution. The seeds were washed with DDW three times andsoaked for three hours in fresh DDW, then plated into 0.5 L containerwith Nitsch medium, containing MS salts, 3% sucrose, Nitsch vitamins and0.8% plant agar (Duchefa, Netherland). The PH was adjusted to 5.8 priorto autoclaving for 20 min at 121° C. 50 seeds where plated on 0.5 Lsterilized container containing the germination medium and left at 25°C. in culture room, 16/8 hrs light/dark cycles, under light intensity of(150 μEm⁻²S). Seedlings where grown for 8 days.

Agrobacterium tumefaciens strain LBA4404 carrying an intact vir regionwhich can mediate the introduction of the T-DNA from the bacteria intoplants. The binary vector plasmids, originated from pPI, were introducedinto the strain LBA 4404 as described above.

For co cultivation, a single colony from freshly streak LB platesupplemented with 300 μg/ml streptomycin and 50 μg/ml kanamycin (Sigma)was used to inoculate 5 ml LB overnight shaking at 28° C. The inoculated5 ml where added to 45 ml LB supplemented with 300 μg/ml streptomycinand 50 μg/ml kanamycin to additional overnight at the same conditions.The overnight culture where centrifuged for 3060 rpm for 10 minutes andrinsed with 50 ml MS medium.

2.5 ml of fine tobacco suspension culture where plated on petri dishes(100×25 mm) containing 50 ml of KCMS murashige minimal organics mediumsupplemented with 0.2 μg/ml 2.4D, kinetin 0.1 μg/ml, thiaminehydrochloride 0.8 μg/ml, potassium acid phosphate 200 μg/ml, biotin 0.5μg/ml, folic acid 0.5 μg/ml, casein hidrolysat 80 μg/ml and plant agar0.8%, PH 5.8 (Duchefa, Netherland).

Whatmann paper filter no. 1 (Whatmann) was autoclaved and placed on topthe of the feeder plates. Any air bubbles and remaining tobaccosuspension media were excluded. The plates were incubated for 24 hoursunder low light conditions (10 μm⁻²S).

8 days old cotyledons were cut at both ends on MS medium and plated for24 hours on the tobacco suspension plates in the same conditions.

The cotyledons were immersed in 5 ml of the rinsed Agrobacterium cellsdiluted in 50 ml MS medium in sterile Petri dish. The concentration ofthe bacteria was ˜5×10⁸ cfu/ml. Following 10 minutes the cotyledons wereblotted carefully to remove any excess of bacterial suspension. 20cotyledons were placed on the feeder plates for 48 hr for co-incubationwith the bacteria under the same conditions.

Cotyledons were then transferred to 2 Zeatin Ribozide (ZR) regenerationmedium (Duchefa, Netherland), containing 400 μg/ml carbenicillin or 150Ticarcillin/potassium clavulanate to inhibit growth of Agrobacterium andKanamycin 100 μg/ml to select for transformed tomato cells.

The cotyledons were transferred to fresh regeneration media after 1month supplemented with 1 ZR 200 carbenicillin and 100 μg/ml Kanamycin.

Shoots where excised when they were approximately 1 cm long andtransferred to 0.5 L containers supplemented with rooting mediacontaining MS medium 50 μg/ml kanamycin, 100 μg/ml carbenicillindisodium, 2 μg/ml IBA (Duchefa, Netherland). After approximately tendays the rooted explants were transferred into soil, under 100%humidity. The humidity was reduced gradually for 24 hours. After 24 hrthe plants were transferred to the greenhouse.

Testing Expression of Foreign Proteins in Trichomes:

A. GUS staining—Gus staining of tomato and tobacco plants was effectedas previously described (15). Briefly: Leaves were fixed in 90% ice coldacetone for 15-20 minutes (on ice), followed by removal of acetone and adouble tissue rinsing with the Working Solution [25 mM Sodium Phosphate(Sigma, USA) buffer pH=7, Ferricyanide (Sigma, USA) 1.25 mM,Ferrocyanide (Sigma, USA) 1.25 mM, Triton X-100 (Sigma, USA) 0.25%, EDTA(BioLab) 0.25 mM] for 15-20 minutes. Rinse solution was removed,replaced with Staining solution [Working solution with5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-GlcA, Duchefa)solubilized in N,N-Dimethylformamide (BioLab) 1.5 mg/ml andDithiothreitol (DTT, Bio Lab) 100 mM] and incubated in the dark (tubeswrapped with aluminum foil) over night at 37° C. Distaining was effectedby sinking the plant tissue in 70% ethanol and heating at 50° C. for ˜2hours. The distaining step was repeated until the plant tissue becametransparent except the blue stained regions. Distained plants werestored in 70% ethanol (BioLab) at room temperature.

ELISA—Human protein detection in plant tissues was effected using HumanInterferon β ELISA Kit (R & D Systems) and Ultra-sensitive Human GrowthHormone ELISA Kit (Diagnostic Systems Laboratories, Inc), according tomanufacture instructions.

Western Blot—Briefly, proteins extracted from the leaves were resolvedon 12% Tris-HCl-Criterion gel (Bio-Rad Laboratories, Inc.) andtransferred by electroporation onto PVDF membranes using the Bio-RadCriterion Precast Gel System (100 V constant voltage at 4° C., 1.5hours). Pre-stained SDS-PAGE standards (Bio-Rad Laboratories, Inc.) wereused as molecular weight markers. Primary antibodies were diluted 1:500and the secondary antibody-HRP conjugate was diluted 1:15,000. Thefollowing anti-recombinant protein polyclonal antibodies were used: goat(Santa Cruz) or sheep (Biosource) polyclonal anti human IFN-β, goatpolyclonal anti human GH (Santa Cruz), rabbit polyclonal anti E.coli-glucuronidase (Molecular Probes). ECL and related reagents wereobtained from Amersham Biosciences.

Bradford protein quantification test—Protein quantification was effectedaccording to Bradford method [Bradford M., Analytical biochemistry, 72:248 (1976)] using Bio-Rad Laboratories, Inc. reagent.

Results

GUS staining was performed on leaves of T1 tomato and tobacco plantstransfected with a binary vector including putative trichome specificpromoters upstream of a GUS gene. Control plants included wild-typetomato and tomato transfected with GUS under the constitutive CaMV S35promoter (FIGS. 1 b-c). As is shown in FIGS. 1 d-h under the regulationof TR2, TR5, TR11, TR25 and TR27 promoters GUS was expressed in atrichome-gland specific manner (FIGS. 1 d-h). A light blue color wasalso found in the stalk cell immediately attached to the gland and innon glandular tissues. Results for tomato T1 generation are summarizedin Table 6, below.

TABLE 6 No of Signal Independent Promoter peptide T1 plants Avg gradeRange 35S No 9 2.1 0-5 TR2 No 2 1.5 0-3 TR5 No 6 0.2 0-1 TR11 No 3 0 0TR11 Stroma 4 0 0 TR11 Lumen 4 0.5 0-1 TR25-2 No 8 0 0 TR25-3 No 18 0.10-1 TR27-P No 5 0.4 0-2

The results presented in Table 6 above indicate that TR2 and TR27-P aremost effective in facilitating expression of heterologous genes in thetomato trichome. These results are of special significance since TR2 hasnever been described before as a trichome active promoter. Furthermore,the addition of a luman directing signal peptide seems to havefacilitated expression in the trichome.

Example 5 Novel Methods for Mechanical Harvesting of Proteins fromTrichomes

Materials and Methods

Harvesting of Trichome proteins:

Four harvesting protocols were attempted to optimize proteinpurification.

Protocol 1—Total trichome protein harvesting was performed by wipingfully expanded leaves with cotton swabs moistened with a solution of 200mM Dithiothreitol (DTT, BioLab). Approximately 5-7 leaves were wiped perswab, with a total of about 1500 leaves. The crude trichome extract wassqueezed from the swab using a syringe, and centrifuged (15000×g for 15minutes at 4° C.). To adsorb phenolic compounds, the supernatant(approximately 70 ml) was treated with Polyvinylpolypyrrolidone (PVPP;Sigma, USA) for 10 minutes on a gentle stirrer followed bycentrifugation (15000 g for 20 minutes) to remove the PVPP. Prior toapplication, PVPP was boiled in 10% HCl for 10 minutes, washedextensively with DDW, air-dried for storage and soaked for at least 3hours in 200 mM DTT solution at 4° C. (ratios were 8-12 g PVPP/250 ml of200 mM DTT, 1-1.5 g/60-85 ml extract). Extracts were concentrated in twosteps of ammonium sulfate precipitation (20%, 75% of saturation). Solidammonium sulfate (0%→20%: 114 g/1 L, 20%→75%: 382 g/1 L) was added tothe extract until the desired concentration was reached while constantstirring. Thereafter the solution was held on ice for about 1 hour withoccasional stirring. The precipitate was collected by centrifugation(15000×g for 20 minutes at 4° C.) and dissolved in 50 mM Tris-HCl, pH 7and 30 mM NaCl.

Protocol 2—Trichome cells and exudates were harvested directly into DDWwhich contained chemicals with antioxidant activity (i.e., citric acid,ascorbic acid or sodium bisulfite). Tomato leaves and shoots were soakedin a container. Gentle shaking of the tissue in the liquid medium causedthe explosion of the glandular trichome cells, type VI and VII (see FIG.1 a) and released the trichome exudates into the media. To eliminate theloss of significant amounts of liquid, tissues were lifted out of themedium and partially dried by shaking it vigorously, letting drops fallback to the container. Trichome yield was measured by inspecting thetreated leaves. Trichome harvesting efficiency was calculated as thepercentage of broken and exploded trichomes, out of total trichomes on agiven leaf area.

Protocol 3—Trichome cells and exudates were harvested directly into aliquid media. Tomato leaves or shoots were put in a container, filledwith tap water. The container was closed and centrifuged at 20 rpm for 5minutes. To eliminate the loss of significant amounts of liquid, tissueswere lifted out of the medium and partially dried by shaking itvigorously, letting drops fall back to the container. Trichome yield wasmeasured by inspecting the treated leaves. Trichome harvestingefficiency was calculated by percentage of broken and explodedtrichomes, out of total trichomes on a given leaf area.

Protocol 4—Trichome cells and exudates were harvested directly into aliquid media. Tomato leaves or shoots were put in a container, filledwith tap water. The container was closed and water was poured on top ofthe leaves using pump which circulated the water in the container.Trichome yield was measured by inspecting the treated leaves. Trichomeharvesting efficiency was calculated by percentage of broken andexploded trichomes, out of total trichomes on a given leaf area.

Results

Protein harvest, purification, yield—Protocol 1 was effected on acommercial tomato variety, grown in a commercial greenhouse, which wasdesigned and used for tomato fruit harvest. Tomato (L. esculentum var591) plants were grown for 3 months. Plant architecture was designed byleaving two main shoots for each plant. Using Bradford analysis it waspossible to calculate total protein yield. Harvesting about 1,100 leavesyielded 16 mg total protein (0.1 mg/ml). Total protein yield wasresolved on Nu-PAGE Novex Bis-Tris gel, 12% (Invitrogen) and proteinbands were visualized by coomassie staining (FIG. 2). A band harboringof an estimated size of 60 kDa was predicted to be the mature PPOprotein according to previous reports (12, 29). PPO is estimated tocount for 60% of total proteins in the trichome.

It will be appreciated that although protocol 1 for protein harvestingis highly effective in collecting most of the trichome exudates, it islabor intensive and slow. The other described methods (2, 3, and 4) weretested to replace the mechanical harvesting step in method 1.

Tomato shoots (1 m long) were dipped in a container and were subjectedto trichome mechanical harvesting using method 2, 3 or 4.

Results, showing the efficiency of each method are presented in theTable 7, below.

TABLE 7 Number Average Range Method of Shoots Efficiency Efficiency 2 398 96-100 3 3 11.3 8-14 4 3 42 38-46 

These results suggest that protocol 2 is the most effective for proteinharvesting from trichomes.

High activity of PPO in trichome may affect protein harvesting andpurification from trichomes. Thus, identification of chemicals which arestrong antioxidants, non toxic, cheap and not affecting proteinstability is mostly desired.

Three compounds were tested: citric acid, ascorbic acid and sodiumbisulfite. Different concentrations of chemicals were used to identifythe chemical with the highest antioxidant activity. Tomato (L.esculentum) young leaves (about 100 mg) were grinded at 4° C. in 200 μlof 10 mM Tris-HCl pH=8 buffer containing appropriate concentration ofantioxidant (0.01-2.0% w/v), incubated for about 2 hrs at 4° C. andcentrifuged at 14,000 rpm for 3 min in order to separate leaves-debrisand liquid fraction. Decreasing of PPO activity was inspected byeliminating the production of brown color of the supernatant as a resultof the oxidation of polyphenols. Table 8 below summarizes the minimalconcentration that enables a decrease of 95% of PPO activity.

TABLE 8 chemical Efficient concentration Citric acid 1.0%  Ascorbic acid1.0%* Sodium bisulfite 0.05%  *After overnight incubation at 4° C. allthe samples undergone browning regardless of the ascorbic acidconcentration.

FIG. 3 shows browning of medium as a result of PPO activity, underdifferent concentrations of Sodium bisulfite.

A buffer without any antioxidant was used as a negative control. DDT wasused as a positive control at the concentration of 200 mM, which isknown to eliminate PPO activity when harvested from trichome cells (12,29).

Example 6 An Automated Machine for Trichome Cells and Trichome ExudateHarvesting

A machine was designed, to enable the automation and up-scaling ofprotein harvesting according to protocol 2. The purpose of the machineis to harvest trichome cells and exudates from full-grown, 3 month oldtomato plants (about 1 meter long and 80 cm in diameter). The machine isbuilt of 2 main parts, (i) 2 m high and 1 m in diameter of cylindershaped container (either made from stainless-steel, glass or a plastic);and (ii) a 2.5 m arm which operates using a 2 speed engine.

The machine has four steps of operation:

1. The plant is tight to the arm manually. The arm is introducing theplant into the container, half full with the liquid medium.

2. The arm is slowly moving up and down (engine is operating on a slowspeed) the plant in the liquid medium such that trichome cells andexudates are released into the medium, without damaging other tissues ofthe plant.

3. The arm is moving the plant just above of the liquid medium.

4. The arm is vigorously shaking the plant up and down (engine isoperating on a high speed). Doing that most of the liquid, whichremained attached to the plant tissues, is released and drops falls backto the container. The semi-dried plant is removed and a new plant istight to the arm.

Example 7 Determining Plant Architecture for Optimizing TrichomeProduction

Typically, the architecture of cultivated tomato plants is designed viabreeding to provide the highest fruit yields, in a given space, in agiven time. Moreover hand labor is routinely being practiced to optimizeplant architecture for that purpose. For trichome optimized expression,plant architecture needs re-design to optimize trichome production, in agiven greenhouse space in a given time. Two approaches for increasingprotein yield were employed essentially, increasing the number oftrichomes in leaves; and increasing the number of leaves on the plant.

Materials Methods and Results

Increasing number of trichomes on leaves—Over 300 tomato cultivars werescreened for trichome density. Leaves of 4 weeks old plants wereinspected, and average trichome density was measured. Best performingcultivars were grown and trichome density was tested again on mature, 14week old plants. Trichome density was compared to previously measureddensity of several tomato lines (1). The seven best performing cultivarswere grown in the next season and trichome density was measured again,to check the heredity of trichome density of two generations. Table 9,below summarizes trichome density of the best performing cultivars.

TABLE 9 Avg. trichome Avg. trichome No. of Plants number of 1^(st) No ofPlants number of 2^(nd) Var 1^(st) generation generation 2^(nd)generation generation 309_2 3 6 3 5.3 305 3 4.7 3 5.3 249_1 3 4.3 3 5.3247 3 3.7 3 6 273_1 3 5.4 3 4.7 294 3 4.5 3 3.2 289 3 4.2 3 3 (Note -the number in the above table represent only the best performingcultivars, out of 300 tested).

Folding a single leaflet and inspecting the edge of the folded leafletwas performed in order to count trichome cells. Trichome number is alltrichomes found on the edge of the leaflet under ×120 magnificationusing binocular microscope (Optika, Italy). Previous publication hascalculated for Lycopersicon hirsutum, var Glabratum (Cultivar No273_(—)1 in this experiment) over 100,000 trichomes per 1 gram of leaf.Assuming trichome density in this experiment remains the same, bestperforming L. esculentum cultivars (No 309_(—)2, 305, 249_(—)1, 247,294, 289), identified here, have the trichome number in the same order.Each leaflet was inspected 3-5 times and an average number wascalculated for the leaflet. Three different leaflet from three differentplants were inspected for each cultivar in each generation.

Four cultivars were identified with the highest density of type VI (FIG.1 a) glandular trichomes on the upper part of the leaves. Among the fivebest performing cultivars, one belongs to Lycopersicon hirsutum, varGlabratum. (273_(—)1) and the rest for cultivated tomato (No 309_(—)2,305, 249_(—)1, 247). Interestingly, the Lycopersicon esculentum speciescultivars exhibited up to 20 fold more coverage of type VI trichomescompared to other cultivars within this species (not shown). Overallbest cultivated cultivars possessed the same density of type VItrichomes, compared to the wild species Lycopersicon hirsutum, varglabratum, which is recognized as the highest trichome density in allLycopersicon genus (1, 16).

Approach B—Increasing number of leaves in a plant—Tomato plantarchitecture was designed manually. 35 days old plants were planted in agreenhouse. Different mechanical treatments were applied to shape plantarchitecture during plant growth. To avoid the collapse of the plantbush, shoots were hanged from the greenhouse ceiling using plasticstrings. The three best performing treatments for plant architecture,aiming to increase trichome yield by increasing the number of leavesproduced are presented hereinbelow:

1. Plant shoot number is not limited, plant height is limited to 1 m,flowers were cut-off before fruit set.

2. Plant shoot number is not limited, plant height is limited to 2 m,flowers were cut-off before fruit set.

3. Plant shoot number is not limited, leading apical meristem was cut(i.e. breaking apical dominance) when reached 0.5 m, flowers werecut-off before fruit set.

4. Plant shoot number was limited to two. Flowers and fruits remaineduntouched (A control treatment, usually applied for greenhouse tomatoes,grown for fruit set).

Three different indeterminant (i.e. the greenhouse type) tomatocultivars (namely 678, 1312, and 2545) previously identified as havinghigh trichome density, were grown in the greenhouse for two months. Thefour above treatments were applied to five plants from each cultivar.Leaf number of each plant was calculated. Table 10, below summarizes theleaf number of tomato plants growing under different mechanical plantdesign.

TABLE 10 Var treat N Rows Mean (No) Std Err (No) 678 1 5 94.2 7.61 678 25 87.4 5.22 678 3 5 71 5.03 678 4 5 27.8 2.75 1312 1 5 125 24.56 1312 25 148.4 13.12 1312 3 5 123 10.22 1312 4 5 54.6 2.84 2545 1 5 95.6 23.892545 2 5 81.4 3.91 2545 3 5 81.8 12.92 2545 4 5 34.4 4.48

As is evident from Table 10, above and FIGS. 5 a-c, a significantincrease [at 0.05 level for cultivar 2545 (FIG. 5 c) and 0.01 level forcultivars 678 and 1312 (FIGS. 5 a-b, respectively)] was observed inresponse to treatments number 1, 2, 3 compared to control (treatment No4). Overall a 50% increase in leaf number (2.25 to 3.39) was observedover the control. Trichome density and PPO enzyme activity in trichomeswere measured in each plant to verify that the increase in leaf numberis not correlated with a decrease in trichome or protein production. Nosignificant change (at 0.05 level) was observed for either trichomenumber or protein accumulation, following growth in leaf number (datanot shown).

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

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What is claimed is:
 1. An isolated polynucleotide comprising the nucleicacid sequence set forth in SEQ ID NO:26.
 2. A nucleic acid constructcomprising the isolated polynucleotide of claim 1 and at least oneheterologous polynucleotide operably linked to the isolatedpolynucleotide.
 3. The nucleic acid construct of claim 2, wherein thenucleic acid construct further comprises; a nucleic acid sequenceencoding a peptide capable of directing transport of a polypeptide fusedthereto into a subcellular compartment of a trichome.
 4. The nucleicacid construct of claim 3, wherein said nucleic acid sequence encodingsaid peptide capable of directing transport of the polypeptide isselected from the group consisting of SEQ ID NOs: 59, 61, 63, 65 and 75.5. The nucleic acid construct of claim 3, wherein said subcellularcompartment of a trichome is a leucoplast.
 6. A transgenic plant celltransformed with the nucleic acid construct of claim
 2. 7. A transgenicplant transformed with the nucleic acid construct of claim
 2. 8. Thenucleic acid construct of claim 2, further comprising an isolatedpolynucleotide comprising a nucleic acid sequence encoding a peptidecapable of directing transport of a polypeptide fused thereto into asubcellular compartment of a trichome, wherein said peptide is encodedby the polynucleotide sequence set forth in SEQ ID NO:59, 61, 63, 65 or75.
 9. A nucleic acid construct comprising the isolated polynucleotideof claim
 8. 10. The nucleic acid construct of claim 9, furthercomprising an expressible polynucleotide sequence translationally fusedto said nucleic acid sequence encoding said peptide.